Long term disease modification using immunostimulatory oligonucleotides

ABSTRACT

The invention provides methods for treating asthma by using multiple rounds of administration of ISS over a period of time to confer long term disease modification.

RELATED APPLICATIONS

This application claims priority benefit of provisional patentapplication No. 61/053,605, filed on May 15, 2008, the disclosure ofwhich is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for treating allergic rhinitis by usingmultiple administrations of one or more immunostimulatory sequences(“ISS”) that confer long term disease modification.

BACKGROUND OF THE INVENTION

The type of immune response generated by infection or other antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. The Th1 subset is responsible forclassical cell-mediated functions such as delayed-type hypersensitivityand activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subsetfunctions more effectively as a helper for B-cell activation. The typeof immune response to an antigen is generally influenced by thecytokines produced by the cells responding to the antigen. Differencesin the cytokines secreted by Th1 and Th2 cells are believed to reflectdifferent biological functions of these two subsets. See, for example,Romagnani (2000) Ann. Allergy Asthma Immunol., 85:9-18.

The Th1 subset may be particularly suited to respond to viralinfections, intracellular pathogens, and tumor cells because it secretesIL-2 and IFN-γ, which activate CTLs. The Th2 subset may be more suitedto respond to free-living bacteria and helminthic parasites and maymediate allergic reactions, since IL-4 and IL-5 are known to induce IgEproduction and eosinophil activation, respectively. In general, Th1 andTh2 cells secrete distinct patterns of cytokines and so one type ofresponse can moderate the activity of the other type of response. Ashift in the Th1/Th2 balance can result in an allergic response, forexample, or, alternatively, in an increased CTL response.

It has been recognized for some time that a Th1-type immune response canbe induced in mammals by administration of certain ISSs. The ISSsinclude sequences referred to as immunostimulatory sequences (“ISS”),often including a CG. See, e.g., PCT Publications WO 98/55495, WO97/28259, U.S. Pat. Nos. 6,194,388; 6,207,646, and 6,498,148; and Krieget al. (1995) Nature, 374:546-49. For many infectious diseases, such astuberculosis and malaria, Th2-type responses are of little protectivevalue against infection. Protein-based vaccines typically induceTh2-type immune responses, characterized by high titers of neutralizingantibodies but without significant cell-mediated immunity. Moreover,some types of antibody responses are inappropriate in certainindications, most notably in allergy where an IgE antibody response canresult in anaphylactic shock.

A treatment for allergic rhinitis that could confer long term benefitshas not been well-characterized either. The invention disclosed hereinprovides teachings useful to address the foregoing.

All patents, patent applications, and publications cited herein arehereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The invention provides for methods for treating allergic rhinitis in anindividual in need thereof by administering to the individual multipleadministrations of an effective amount of an ISS. The ISS may beadministered with an allergen. In one embodiment, the allergen ispresent in the environment naturally. In another embodiment, theallergen is a seasonal allergen. Any ISS may be used to effect thetreatment. In one aspect, multiple administrations of ISS can conferlong term disease modification. In one embodiment, the diseasemodification is a suppression of Th2 immune response in the individualto whom the ISS was administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that depicts results for six genes ISS important forthe development of a Th2-type airway inflammatory response whensensitized mice are challenged intranasally with ragweed. The data areexpressed as gene/ubiquitin ratio.

FIG. 2 is a graph that depicts inhibition of Th2 gene induction forGOB-5 and C2 by pretreatment with 1018 ISS. The data are expressed asgene/ubiquitin ratio.

FIG. 3 is a graph that depicts levels of the Th2-type cytokines IL-4 andIL-13 detected in BAL fluid. The double asterisks indicate a statisticalsignificance of p<0.01. The single asterisk indicates a statisticalsignificance of p<0.05.

FIG. 4 is a graph that depicts levels of the Th2-type cytokine IL-10 andTh1 type cytokine INF-γ detected in BAL fluid. The double asterisksindicate a statistical significance of p<0.01.

FIG. 5 is a graph that depicts absolute number of eosinophils detectedin BAL fluid. The double asterisks indicate a statistical significanceof p<0.01.

FIG. 6 is a graph that depicts levels of the Th2-type cytokines IL-4,IL-5, IL-10 and IL-13 measured in lavage fluid (BAL fluid) in mice thathave been treated with multiple administrations of 1018 ISS.

FIG. 7 is a graph that depicts levels of the Th2-type cytokines IL-4,IL-5, IL-10 and IL-13 measured in lavage fluid (BAL fluid) in mice thathave been treated with multiple administrations of 1018 ISS or TOLAMBA.

FIG. 8 is a graph that depicts results that show that re-sensitizationwith ragweed or ovalbumin does not lead to allergen-induced airwayeosinophilia.

FIG. 9 is a graph that depicts results that show that re-sensitizationwith ragweed or ovalbumin does not lead to allergen-induced elevated balth2 cytokines.

FIG. 10 is a graph that depicts results that show that presence ofova-specific IgE indicates successful system sensitization to ovalbumin.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein provides method for treating allergic rhinitis inan individual by administering an effective amount of animmunostimulatory sequence (ISS) over multiple administrations to theindividual. In one aspect of the invention, long term diseasemodification can be conferred by using multiple administrations of ISS.Long term disease modification includes the suppression of a Th2response in the individual. In some cases, the suppression is aninhibition of a Th2 response.

General Methods

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989) and Molecular Cloning: A Laboratory Manual, third edition(Sambrook and Russel, 2001), (jointly and individually referred toherein as “Sambrook”); Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook ofExperimental Immunology (D. M. Weir & C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,1987); Current Protocols in Molecular Biology (F. M. Ausubel et al.,eds., 1987, including supplements through 2001); PCR: The PolymeraseChain Reaction, (Mullis et al., eds., 1994); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); The Immunoassay Handbook(D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (GregT. Hermanson, ed., Academic Press, 1996); Methods of ImmunologicalAnalysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim:VCH Verlags gesellschaft mbH, 1993), Harlow and Lane (1988) Antibodies,A Laboratory Manual, Cold Spring Harbor Publications, New York, andHarlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (jointly andindividually referred to herein as “Harlow and Lane”), Beaucage et al.eds., Current Protocols in Nucleic Acid Chemistry (John Wiley & Sons,Inc., New York, 2000); and Agrawal, ed., Protocols for Oligonucleotidesand Analogs, Synthesis and Properties (Humana Press Inc., New Jersey,1993).

DEFINITIONS

As used herein, the term “allergen” means an antigen or antigenicportion of a molecule, usually a protein, which elicits an allergicresponse upon exposure to a subject. Typically the subject is allergicto the allergen as indicated, for instance, by the wheal and flare testor any method known in the art. A molecule is said to be an allergeneven if only a small subset of subjects exhibit an allergic immuneresponse (e.g., IgE) upon exposure to the molecule. An allergen may bepresent in the environment in minute quantities or in larger quantitiesdepending on the season. Examples of allergens are listed in Table 1infra.

An “individual” is a vertebrate, such as mouse, and is preferably amammal, more preferably a human. Mammals include, but are not limitedto, humans, primates, farm animals, sport animals, rodents and pets.

An “effective amount” of a substance is that amount sufficient to effectbeneficial or desired results, including clinical results, and, as such,an “effective amount” depends upon the context in which it is beingapplied. In the context of administering a composition that modulates animmune response, either with or without a co-administered antigen, aneffective amount of an ISS (and antigen, if applicable) is an amountsufficient to achieve such a modulation as compared to the immuneresponse obtained when the antigen is administered alone. An effectiveamount can confer long term benefits of disease modification, such assuppression and/or inhibition of Th2 immune response. An effectiveamount can be administered in one or more administrations.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation or amelioration ofone or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, and/or amelioration or palliation of the disease state.“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment.

As used herein, the term “long term disease modification” refers to thereduction or elimination of one or more allergic rhinitis symptoms for aperiod of at least 3 weeks following administration of the last dose ofISS, preferably for a period of at least 8 weeks, most preferably for aperiod of at least 12 weeks. The allergic rhinitis symptoms include, butare not limited to, nasal symptoms (rhinorrhea, congestion, excess nasalsecretion/runny nose, sneezing, itching) and non-nasal symptoms(itchy/gritty eyes, tearing, watery eyes, red or burning eyes,post-nasal drip, ear or palate itching).

Biological Effects of ISS

It been observed that long term disease modification can be conferred onindividuals with allergic rhinitis by using multiple administrations ofISS. The examples provide some illustration of this observation. Example1 discloses that the direct effects of one type of ISS, the 1018 ISS forexample, lasts about one week in a murine model of allergic asthma.Examples 2 and 3 illustrate that the Th2 response can be suppressed inindividuals to whom 1018 ISS has been multiply administered for at least8 weeks. The long term effects of Th2 suppression can last at least 13weeks. Thus, in one aspect, the invention provides for treating allergicrhinitis long term by administering to an individual an effective amountof ISS for at least 8 weeks. This long term effect of the treatment canlast at least 13 weeks. The invention contemplates methods for providingthe long term benefits for individuals who may have allergic rhinitis byusing multiple administration of ISS for at least 8 weeks to confer longterm disease modification that persists for at least 13, 15, 17, 19, 21or 25 weeks.

Functionally, ISS enhance the cellular and humoral immune responses inan individual, particularly lymphocyte proliferation and the release ofcytokines (including interferon or IFN) by individual monocytes andnatural killer (NK) cells. Immunostimulation by synthetic ISS in vivooccurs by contacting individual lymphocytes with, for example ISS, ISSoligonucleotide conjugates and ISS-containing recombinant expressionvectors. See, for example, U.S. Pat. No. 6,610,661 and WO 97/28259.Thus, while native microbial ISS stimulate the individual immune systemto respond to infection, synthetic analogs of these ISS are usefultherapeutically to modulate the individual immune response not only tomicrobial antigens, but also to allergens and other substances.

ISS Compositions

The method of this invention can be practiced by using any type of ISS.In one embodiment, the 1018 ISS is used. The structure of 1018 ISS hasbeen published in multiple scientific articles as well as patents. See,for example, Hessel et al. (2005) J. Exp. Med., 202(11):1563. Ingeneral, 1018 ISS is (5′-TGACTGTGAACGTTCGAGATGA-3′). In anotherembodiment, one or more ISS containing CpG motif(s) can be used. See,for example, U.S. Publication No. 2006/0058254 or WO 2004/058179. Inanother embodiment, one or more chimeric immunomodulatory compound(“CIC”) can be used. See, for example, U.S. Publication No.2004/0132677.

In accordance with the present invention, the ISS contains at least onepalindromic sequence (i.e., palindrome) of at least 8 bases in lengthcontaining at least one CG dinucleotide. The ISS also contains at leastone TCG trinucleotide sequence at or near the 5′ end of thepolynucleotide (i.e., 5′-TCG). In some instances, the palindromicsequence and the 5′-TCG are separated by 0, 1 or 2 bases in the ISS. Insome instances the palindromic sequence includes all or part of the5′-TCG.

ISSs have been described in the art and their activity may be readilyidentified using standard assays which indicate various aspects of theimmune response, such as cytokine secretion, antibody production, NKcell activation, B cell proliferation, T cell proliferation. See, e.g.,WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995) Nature374:546-549; Yamamoto et al. (1992a); Ballas et al. (1996); Klinman etal. (1997); Sato et al. (1996); Pisetsky (1996a); Shimada et al. (1986)Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J. Immunol.156:4570-4575; Roman et al. (1997); Lipford et al. (1997a); WO 98/55495and WO 00/61151. Accordingly, these and other methods can be used toidentify, test and/or confirm immunomodulatory ISSs.

The ISS can be of any length greater than 10 bases or base pairs,preferably greater than 15 bases or base pairs, more preferably greaterthan 20 bases or base pairs in length. It is understood that, withrespect to formulae described herein, any and all parameters areindependently selected. For example, if x=0-2, y may be independentlyselected regardless of the values of x (or any other selectableparameter in a formula).

In some embodiments, an ISS comprises (a) a palindromic sequence atleast 8 bases in length which contains at least two CG dinucleotides,where the CG dinucleotides are separated from each other by 0, 1, 2, 3,4 or 5 bases, and (b) a (TCG)_(y) sequence positioned 0, 1, 2, or 3bases from the 5′ end of the polynucleotide, where y is 1 or 2, andwhere the 3′ end of the (TCG)_(y) sequence is separated from the 5′ endof the palindromic sequence by 0, 1 or 2 bases. In some embodiments, aCG dinucleotide of the (TCG)_(y) sequence of (b) may count for one ofthe at least two CG dinucleotides in the palindromic sequence of (a). Insome embodiments, the CG dinucleotides of the palindromic sequence areseparated from each other by 1, 3 or 4 bases. In some ISSs of theinvention, whether described in this paragraph or elsewhere in theapplication, the palindromic sequence has a base composition of lessthan two-thirds G's and C's. In some embodiments, the palindromicsequence has a base composition of greater than one-third A's and T's.

In some embodiments, an ISS comprises (a) a palindromic sequence atleast 8 bases in length which contains at least two CG dinucleotides,where the CG dinucleotides are separated from each other by 0, 1, 2, 3,4 or 5 bases, and (b) a (TCG)_(y) sequence positioned 0, 1, 2, or 3bases from the 5′ end of the polynucleotide, where y is 1 or 2, wherethe palindromic sequence includes all or part of the (TCG)_(y) sequence,and where a CG dinucleotide of the (TCG)_(y) sequence of (b) may countfor one of the CG dinucleotides of the palindromic sequence of (a).Preferably, in some embodiments, the CG dinucleotides of the palindromicsequence are separated from each other by 1, 3 or 4 bases.

Accordingly, in some embodiments, an ISS may comprise a sequence of theformula: 5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁CGX₁′(CG)_(p))_(z) wherein Nare nucleosides with x=0-3, y=1-4, w=−1, 0, 1 or 2, p=0 or 1, q=0, 1 or2, and z=1-20, wherein X₁ and X₁′ are self-complimentary and wherein the5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end ofthe polynucleotide. The ISS further comprises a palindromic sequence 8bases in length or greater wherein the palindromic sequence comprises atleast one of the (X₁CGX₁′(CG)_(p)) sequences. In an ISS with w=−1, the3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of the first(X₁CGX₁′(CG)_(p)) sequence. In some embodiments, the (TCG(N_(q)))_(y)sequence is separated from the palindromic sequence by 0, 1 or 2 bases.In other embodiments, the palindromic sequence includes all or part ofthe (TCG(N_(q)))_(y) sequence. In some embodiments, when p=0, X₁ iseither A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p))_(z) wherein Nare nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2, p=0 or 1,q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′, and X₃ and X₃′are self-complimentary and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The ISSfurther comprises a palindromic sequence 8 bases in length or greaterwherein the palindromic sequence comprises the first (X₁X₂X₃CGX₃′X₂′X₁′)of the at least one (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) sequence. In an ISS withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂,respectively, of the first (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) sequence. In anISS with w=−3, the antepenultimate (i.e., third to last), thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, and X₃,respectively, of the first (X₁X₂X₃CGX₃′X₂′X₁′(CG)_(p)) sequence. In someembodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, when p=1, X₁, X₂, and X₃ are each eitherA or T. In some embodiments, when p=0, at least two of X₁, X₂, and X₃are either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p))_(z)wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2,p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′, X₃and X₃′, and X₄ and X₄′ are self-complimentary and wherein the 5′ T ofthe (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The ISS further comprises a palindromic sequence 10bases in length or greater wherein the palindromic sequence comprisesthe first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′) of the at least one(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−1, the 3′base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of the first(X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂,respectively, of the first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) sequence. Inan ISS with w=−3, the antepenultimate (i.e., third to last), thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, and X₃,respectively, of the first (X₁X₂X₃X₄CGX₄′X₃′X₂′X₁′(CG)_(p)) sequence. Insome embodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, when p=1, at least three of X₁, X₂, X₃,and X₄ are either A or T. In some embodiments, when p=0, at least two ofX₁, X₂, X₃, and X₄ are either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁CGCGX₁′(CG)_(p))_(z) wherein N arenucleosides with x=0-3, y=1-4, w=−1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2,and z=1-20, wherein X₁ and X₁′ are self-complimentary and wherein the 5′T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The ISS further comprises a palindromic sequence 8 basesin length or greater wherein the palindromic sequence comprises thefirst (X₁CGCGX₁′) of the at least one (X₁CGCGX₁′(CG)_(p)) sequence. Inan ISS with w=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′X₁ of the first (X₁CGCGX₁′(CG)_(p)) sequence. In some embodiments, the(TCG(N_(q)))_(y) sequence is separated from the palindromic sequence by0, 1 or 2 bases. In other embodiments, the palindromic sequence includesall or part of the (TCG(N_(q)))_(y) sequence.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q))_(y)N_(w)(CGX₁X₁′CG(CG)_(p))_(z) wherein N arenucleosides with x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0 or 1, q=0, 1 or 2,and z=1-20, wherein X₁ and X₁′ are self-complimentary and wherein the 5′T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ end of thepolynucleotide. The ISS further comprises a palindromic sequence 8 basesin length or greater wherein the palindromic sequence comprises thefirst (CGX₁X₁′CG) of the at least one (CGX₁X₁′CG(CG)_(p)) sequence. Inan ISS with w=−2, the penultimate (i.e., second to last) and theultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequence are CGand are the 5′ CG of the first (CGX₁X₁′CG(CG)_(p)) sequence. In someembodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p))_(z) wherein Nare nucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′, and X₃ and X₃′ areself-complimentary and wherein the 5′ T of the (TCG(N_(q)))_(y) sequenceis 0-3 bases from the 5′ end of the polynucleotide. The ISS furthercomprises a palindromic sequence 10 bases in length or greater whereinthe palindromic sequence comprises the first (X₁X₂CGX₃X₃′CGX₂′X₁′) ofthe at least one (X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p)) sequence. In an ISS withw=−1, the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of thefirst (X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂,respectively, of the first (X₁X₂CGX₃X₃′CGX₂′X₁′(CG)_(p)) sequence. Insome embodiments, the (TCG(N_(q)))_(y) sequence is separated from thepalindromic sequence by 0, 1 or 2 bases. In other embodiments, thepalindromic sequence includes all or part of the (TCG(N_(q)))_(y)sequence. In some embodiments, when p=1, X₁, X₂, and X₃ are each eitherA or T. In some embodiments, when p=0, at least two of X₁, X₂, and X₃are either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q))_(y)N_(w)(X₁X₂CGX₂′X₁′(CG)_(p))_(z) wherein N arenucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′ are self-complimentary,and wherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases fromthe 5′ end of the polynucleotide. The ISS further comprises apalindromic sequence 8 bases in length or greater wherein thepalindromic sequence comprises the first (X₁X₂CGX₂′X₁′) of the at leastone (X₁X₂CGX₂′X₁′(CG)_(p))_(z) sequence. In an ISS with w=−1, the 3′base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of the first(X₁X₂CGX₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, the penultimate(i.e., second to last) and the ultimate (i.e., last) 3′ bases of the(TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂, respectively, of thefirst (X₁X₂CGX₂′X₁′(CG)_(p)) sequence. In some embodiments, the(TCG(N_(q)))_(y) sequence is separated from the palindromic sequence by0, 1 or 2 bases. In other embodiments, the palindromic sequence includesall or part of the (TCG(N_(q)))_(y) sequence. In some embodiments, X₁and X₂ are each either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p))_(z)wherein N are nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2,p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′, X₃and X₃′, X₄ and X₄′, and X₅ and X₅′ are self-complimentary, and whereinthe 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases from the 5′ endof the polynucleotide. The ISS further comprises a palindromic sequence12 bases in length or greater wherein the palindromic sequence comprisesthe first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′) of the at least one((X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−1,the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of the first(X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂,respectively, of the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p))sequence. In an ISS with w=−3, the antepenultimate (i.e., third tolast), the penultimate (i.e., second to last) and the ultimate (i.e.,last) 3′ bases of the (TCG(N_(q)))_(y) sequence are the 5′ X₁, X₂, andX₃, respectively, of the first (X₁X₂X₃X₄X₅CGX₅′X₄′X₃′X₂′X₁′(CG)_(p))sequence. In some embodiments, the (TCG(N_(q)))_(y) sequence isseparated from the palindromic sequence by 0, 1 or 2 bases. In otherembodiments, the palindromic sequence includes all or part of the(TCG(N_(q)))_(y) sequence. In some embodiments, at least three of X₁,X₂, X₃, X₄, and X₅ are either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q))_(y)N_(w)(X₁X₂CGCGX₂′X₁′(CG)_(p))_(z) wherein N arenucleosides with x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or2, and z=1-20, wherein X₁ and X₁′, and X₂ and X₂′ areself-complimentary, and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The ISSfurther comprises a palindromic sequence 8 bases in length or greaterwherein the palindromic sequence comprises the first (X₁X₂CGCGX₂′X₁′) ofthe at least one (X₁X₂CGCGX₂′X₁′(CG)_(p)) sequence. In an ISS with w=−1,the 3′ base of the (TCG(N_(q)))_(y) sequence is the 5′ X₁ of the first(X₁X₂CGCGX₂′X₁′(CG)_(p)) sequence. In an ISS with w=−2, the penultimate(i.e., second to last) and the ultimate (i.e., last) 3′ bases of the(TCG(N_(q)))_(y) sequence are the 5′ X₁ and X₂, respectively, of thefirst (X₁X₂CGCGX₂′X₁′(CG)_(p)) sequence. In some embodiments, the(TCG(N_(q)))_(y) sequence is separated from the palindromic sequence by0, 1 or 2 bases. In other embodiments, the palindromic sequence includesall or part of the (TCG(N_(q)))_(y) sequence. In some embodiments, X₁and X₂ are each either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q)))_(y)N_(w)(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p))_(z) wherein Nare nucleosides with x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2, p=0 or 1,q=0, 1 or 2, and z=1-20, wherein X₁ and X₁′, X₂ and X₂′ and X₃ and X₃′are self-complimentary, and wherein the 5′ T of the (TCG(N_(q)))_(y)sequence is 0-3 bases from the 5′ end of the polynucleotide. The ISSfurther comprises a palindromic sequence 10 bases in length or greaterwherein the palindromic sequence comprises the first(X₁X₂X₃CGCGX₃′X₂′X₁′) of the at least one (X₁X₂X₃CGCGX₃′X₂X₁′(CG)_(p))sequence. In an ISS with w=−1, the 3′ base of the (TCG(N_(q)))_(y)sequence is the 5′ X₁ of the first (X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p))sequence. In an ISS with w=−2, the penultimate (i.e., second to last)and the ultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y) sequenceare the 5′ X₁ and X₂, respectively, of the first(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p)) sequence. In an ISS with w=−3, theantepenultimate (i.e., third to last), the penultimate (i.e., second tolast) and the ultimate (i.e., last) 3′ bases of the (TCG(N_(q)))_(y)sequence are the 5′ X₁, X₂, and X₃, respectively, of the first(X₁X₂X₃CGCGX₃′X₂′X₁′(CG)_(p)) sequence. In some embodiments, the(TCG(N_(q)))_(y) sequence is separated from the palindromic sequence by0, 1 or 2 bases. In other embodiments, the palindromic sequence includesall or part of the (TCG(N_(q)))_(y) sequence. In some embodiments, whenp=1, X₁, X₂, and X₃ are each either A or T. In some embodiments, whenp=0, at least two of X₁, X₂, and X₃ are either A or T.

In some embodiments, an ISS may comprise a sequence of the formula:5′-N_(x)(TCG(N_(q))_(y)N_(w)(CGX₁X₂X₂′X₁′CG(CG)_(p))_(z) wherein N arenucleosides with x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0 or 1, q=0, 1 or 2,and z=1-20, wherein X₁ and X₁′, and X₂ and X₂′ are self-complimentary,and wherein the 5′ T of the (TCG(N_(q)))_(y) sequence is 0-3 bases fromthe 5′ end of the polynucleotide. The ISS further comprises apalindromic sequence 8 bases in length or greater wherein thepalindromic sequence comprises the first (CGX₁X₂X₂′X₁′CG) of the atleast one (CGX₁X₂X₂′X₁′CG(CG)_(p)) sequence. In an ISS with w=−2, thepenultimate (i.e., second to last) and the ultimate (i.e., last) 3′bases of the (TCG(N_(q)))_(y) sequence are CG and are the 5′ CG of thefirst (CGX₁X₂X₂′X₁′CG(CG)_(p)) sequence. In some embodiments, the(TCG(N_(q)))_(y) sequence is separated from the palindromic sequence by0, 1 or 2 bases. In other embodiments, the palindromic sequence includesall or part of the (TCG(N_(q)))_(y) sequence. In some embodiments, X₁and X₂ are each either A or T.

For ISSs comprising any of the motifs described herein where y=2 ormore, the (N_(q)) in each of the y repetitions of the (TCG(N_(q))) isindependently selected. For example, in an ISS with y=2, the firstTCG(N_(q)) may have N=A and q=1 and the second TCG(N_(q)) may have q=0in which case this portion of the ISS would be . . . TCGATCG . . . . Insome embodiments of ISSs comprising any of the motifs described hereinin some embodiments, x is preferably 0 or 1. In some embodiments of ISSscomprising any of the motifs described herein, y is preferably 1 or 2.In some embodiments of ISSs comprising any of the motifs describedherein, w is preferably 0. In some embodiments of ISSs comprising any ofthe motifs described herein, z is preferably 1, 2, 3, 4, 5, 6, 7 or 8.

As noted above, the ISSs contain at least one the palindromic sequenceat least 8 bases in length. In some embodiments, an ISS contains atleast one palindromic sequence of at least the following lengths (inbases): 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30. In some embodiments,the palindromic sequence is repeated at least once in an ISS. In someembodiments, the palindromic sequence also includes bases 5′ of the(TCG(N_(q)))_(y) sequence, if any.

Non-limiting examples of specific ISSs that can be used in accordancewith the teachings above can be found in U.S. Publication No.2006/0058254 and also in U.S. Publication No. 2004/0132677.

Modifications to ISS

An ISS may contain modifications. Modifications of ISS include any knownin the art, but are not limited to, modifications of the 3′OH or 5′OHgroup, modifications of the nucleotide base, modifications of the sugarcomponent, and modifications of the phosphate group. Modified bases maybe included in the palindromic sequence of an ISS as long as themodified base(s) maintains the same specificity for its naturalcomplement through Watson-Crick base pairing (e.g., the palindromicportion of the ISS is still self-complementary).

An ISS may contain naturally-occurring or modified, non-naturallyoccurring bases, and may contain modified sugar, phosphate, and/ortermini. For example, in addition to phosphodiester linkages, phosphatemodifications include, but are not limited to, methyl phosphonate,phosphorothioate, phosphoramidate (bridging or non-bridging),phosphotriester and phosphorodithioate and may be used in anycombination. Other non-phosphate linkages may also be used. In someembodiments, polynucleotides of the present invention comprise onlyphosphorothioate backbones. In some embodiments, polynucleotides of thepresent invention comprise only phosphodiester backbones. In someembodiments, an ISS may comprise a combination of phosphate linkages inthe phosphate backbone such as a combination of phosphodiester andphosphorothioate linkages.

Sugar modifications known in the field, such as 2′-alkoxy-RNA analogs,2′-amino-RNA analogs, 2′-fluoro-DNA, and 2′-alkoxy- or amino-RNA/DNAchimeras and others described herein, may also be made and combined withany phosphate modification. Examples of base modifications include, butare not limited to, addition of an electron-withdrawing moiety to C-5and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine,5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) and C-5 and/or C-6of a uracil of the ISS (e.g., 5-bromouracil, 5-chlorouracil,5-fluorouracil, 5-iodouracil). See, for example, WO 99/62923. The use ofa base modification in a palindromic sequence of an ISS should notinterfere with the self-complimentary ability of the bases involved forWatson-Crick base pairing. However, outside of a palindromic sequence,modified bases may be used without this restriction.

In addition, backbone phosphate group modifications (e.g.,methylphosphonate, phosphorothioate, phosphoroamidate andphosphorodithioate internucleotide linkages) can confer immunomodulatoryactivity on the ISS and enhance their stability in vivo, making themparticularly useful in therapeutic applications. A particularly usefulphosphate group modification is the conversion to the phosphorothioateor phosphorodithioate forms of the ISS oligonucleotides. In addition totheir potentially immunomodulatory properties, phosphorothioates andphosphorodithioates are more resistant to degradation in vivo than theirunmodified oligonucleotide counterparts, making the ISS of the inventionmore available to the individual.

Synthesis of and Screening for ISS

The ISS can be synthesized using techniques and nucleic acid synthesisequipment which are well known in the art including, but not limited to,enzymatic methods, chemical methods, and the degradation of largeroligonucleotide sequences. See, for example, Ausubel et al. (1987) andSambrook et al. (1989). When assembled enzymatically, the individualunits can be ligated, for example, with a ligase such as T4 DNA or RNAligase. See, for example, U.S. Pat. No. 5,124,246. Oligonucleotidedegradation can be accomplished through the exposure of anoligonucleotide to a nuclease, as exemplified in U.S. Pat. No.4,650,675.

The ISS can also be isolated using conventional polynucleotide isolationprocedures. Such procedures include, but are not limited to,hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences, antibody screening of expression libraries todetect shared structural features and synthesis of particular nativesequences by the polymerase chain reaction.

Circular ISS can be isolated, synthesized through recombinant methods,or chemically synthesized. Where the circular ISS is obtained throughisolation or through recombinant methods, the ISS will preferably be aplasmid. The chemical synthesis of smaller circular oligonucleotides canbe performed using any method described in the literature. See, forinstance, Gao et al. (1995) Nucleic Acids Res. 23:2025-2029; and Wang etal. (1994) Nucleic Acids Res. 22:2326-2333.

Duplex (i.e., double stranded) and hairpin forms of most ISSs are indynamic equilibrium, with the hairpin form generally favored at lowpolynucleotide concentration and higher temperatures. Covalentinterstrand or intrastrand cross-links increases duplex or hairpinstability, respectively, towards thermal-, ionic-, pH-, andconcentration-induced conformational changes. Chemical cross-links canbe used to lock the polynucleotide into either the duplex or the hairpinform for physicochemical and biological characterization. Cross-linkedISSs that are conformationally homogeneous and are “locked” in theirmost active form (either duplex or hairpin form) could potentially bemore active than their uncross-linked counterparts. Accordingly, someISSs of the invention contain covalent interstrand and/or intrastrandcross-links.

A variety of ways to chemically cross-link duplex DNA are known in theart. Any cross-linking method may be used as long as the cross-linkedpolynucleotide product possesses the desired immunomodulatory activity.

One method, for example, results in a disulfide bridge between twoopposing thymidines at the terminus of the duplex or hairpin. For thiscross-linking method, the oligonucleotide(s) of interest is synthesizedwith a 5′-DMT-N3-(tBu-SS-ethyl)thymidine-3′-phosphoramidite (“T*”). Toform the disulfide bridge, the mixed disulfide bonds are reduced,oligonucleotide purified, the strands hybridized and the compoundair-oxidized to form the intrastrand cross-link in the case of a hairpinform or the interstrand cross-link in the case of a duplex form.Alternatively, the oligonucleotides may be hybridized first and thenreduced, purified and air-oxidized. Such methods and others aredescribed, for example, in Glick et al. (1991) J. Org. Chem.56:6746-6747, Glick et al. (1992) J. Am. Chem. Soc. 114:5447-5448,Goodwin et al. (1994) Tetrahedron Letters 35:1647-1650, Wang et al.(1995) J. Am. Chem. Soc. 117:2981-2991, Osborne et al. (1996) Bioorganic& Medicinal Chemistry Letters 6:2339-2342 and Osborne et al. (1996) J.Am. Chem. Soc. 118:11993-12003.

Another cross-linking method forms a disulfide bridge between offsetresidues in the duplex or hairpin structure. For this cross-linkingmethod, the oligonucleotide(s) of interest is synthesized withconvertible nucleosides (commercially available, for example, from GlenResearch). This method utilizes, for example, an A-A disulfide or a C-Adisulfide bridge and linkages through other bases are also possible. Toform the disulfide-modified polynucleotide, the polynucleotidecontaining the convertible nucleoside is reacted with cystamine (orother disulfide-containing amine). To form the disulfide bridge, themixed disulfide bonds are reduced, oligonucleotide purified, the strandshybridized and the compound air-oxidized to form the intrastrandcross-link in the case of a hairpin form or the interstrand cross-linkin the case of a duplex form. Alternatively, the oligonucleotides may behybridized first and then reduced, purified and air-oxidized. Suchmethods and others are described, for example, in Glick et al. (1991) J.Org. Chem. 56:6746-6747, Glick et al. (1992) J. Am. Chem. Soc.114:5447-5448, Goodwin et al. (1994) Tetrahedron Letters 35:1647-1650,Wang et al. (1995) J. Am. Chem. Soc. 117:2981-2991, Osborne et al.(1996) Bioorganic & Medicinal Chemistry Letters 6:2339-2342 and Osborneet al. (1996) J. Am. Chem. Soc. 118:11993-12003.

Another cross-linking method forms a disulfide bridge between offsetresidues in the duplex or hairpin structure. For this cross-linkingmethod, the oligonucleotide(s) of interest is synthesized withconvertible nucleosides (commercially available, for example, from GlenResearch). This method utilizes, for example, an A-A disulfide or a C-Adisulfide bridge and linkages through other bases are also possible. Toform the disulfide-modified polynucleotide, the polynucleotidecontaining the convertible nucleoside is reacted with cystamine (orother disulfide-containing amine). To form the disulfide bridge, themixed disulfide bonds are reduced, oligonucleotide purified, the strandshybridized and the compound air-oxidized to form the intrastrandcross-link in the case of a hairpin form or the interstrand cross-linkin the case of a duplex form. Alternatively, the oligonucleotides may behybridized first and then reduced, purified and air-oxidized. Suchmethods are described, for example, in Ferentz et al. (1991) J. Am.Chem. Soc. 113:4000-4002 and Ferentz et al. (1993) J. Am. Chem. Soc.115:9006-9014.

The techniques for making polynucleotides and modified polynucleotidesare known in the art. Naturally occurring DNA or RNA, containingphosphodiester linkages, is generally synthesized by sequentiallycoupling the appropriate nucleoside phosphoramidite to the 5′-hydroxygroup of the growing oligonucleotide attached to a solid support at the3′-end, followed by oxidation of the intermediate phosphite triester toa phosphate triester. Once the desired polynucleotide sequence has beensynthesized, the polynucleotide is removed from the support, thephosphate triester groups are deprotected to phosphate diesters and thenucleoside bases are deprotected using aqueous ammonia or other bases.See, for example, Beaucage (1993) “Oligodeoxyribonucleotide Synthesis”in Protocols for Oligonucleotides and Analogs, Synthesis and Properties(Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al. (1984) DNA3:401 and U.S. Pat. No. 4,458,066.

The ISS can also contain phosphate-modified polynucleotides, some ofwhich are known to stabilize the polynucleotide. Accordingly, someembodiments includes stabilized ISSs. Synthesis of polynucleotidescontaining modified phosphate linkages or non-phosphate linkages is alsoknown in the art. For a review, see Matteucci (1997) “OligonucleotideAnalogs: an Overview” in Oligonucleotides as Therapeutic Agents, (D. J.Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, N.Y. Thephosphorous derivative (or modified phosphate group) which can beattached to the sugar or sugar analog moiety in the polynucleotides ofthe present invention can be a monophosphate, diphosphate, triphosphate,alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidateor the like. The preparation of the above-noted phosphate analogs, andtheir incorporation into nucleotides, modified nucleotides andoligonucleotides, per se, is also known and need not be described herein detail. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-1848;Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz etal. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis ofphosphorothioate oligonucleotides is similar to that described above fornaturally occurring oligonucleotides except that the oxidation step isreplaced by a sulfurization step (Zon (1993) “OligonucleosidePhosphorothioates” in Protocols for Oligonucleotides and Analogs,Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).Similarly the synthesis of other phosphate analogs, such asphosphotriester (Miller et al. (1971) JACS 93:6657-6665), non-bridgingphosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3′ to P5′phosphoramidiates (Nelson et al. (1997) JOC 62:7278-7287) andphosphorodithioates (U.S. Pat. No. 5,453,496) has also been described.Other non-phosphorous based modified oligonucleotides can also be used(Stirchak et al. (1989) Nucleic Acids Res. 17:6129-6141).Polynucleotides with phosphorothioate backbones can be more immunogenicthan those with phosphodiester backbones and appear to be more resistantto degradation after injection into the host. Braun et al. (1988) J.Immunol. 141:2084-2089; and Latimer et al. (1995) Mol. Immunol.32:1057-1064.

ISSs used in the invention can comprise one or more ribonucleotides(containing ribose as the only or principal sugar component),deoxyribonucleotides (containing deoxyribose as the principal sugarcomponent), or, as is known in the art, modified sugars or sugar analogscan be incorporated in the ISS. Thus, in addition to ribose anddeoxyribose, the sugar moiety can be pentose, deoxypentose, hexose,deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar “analog”cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl form.In the ISS, the sugar moiety is preferably the furanoside of ribose,deoxyribose, arabinose or 2′-O-alkylribose, and the sugar can beattached to the respective heterocyclic bases either in α or β anomericconfiguration. Sugar modifications include, but are not limited to,2′-alkoxy-RNA analogs, 2′-amino-RNA analogs, 2′-fluoro-DNA, and2′-alkoxy- or amino-RNA/DNA chimeras. For example, a sugar modificationin the ISS includes, but is not limited to, 2′-O-methyl-uridine and2′-O-methyl-cytidine. The preparation of these sugars or sugar analogsand the respective “nucleosides” wherein such sugars or analogs areattached to a heterocyclic base (nucleic acid base) per se is known, andneed not be described here, except to the extent such preparation canpertain to any specific example. Sugar modifications may also be madeand combined with any phosphate modification in the preparation of anISS.

The heterocyclic bases, or nucleic acid bases, which are incorporated inthe ISS can be the naturally-occurring principal purine and pyrimidinebases, (namely uracil, thymine, cytosine, adenine and guanine, asmentioned above), as well as naturally-occurring and syntheticmodifications of said principal bases. Thus, an ISS may include2′-deoxyuridine and/or 2-amino-2′-deoxyadenosine.

Those skilled in the art will recognize that a large number of“synthetic” non-natural nucleosides comprising various heterocyclicbases and various sugar moieties (and sugar analogs) are available inthe art, and that as long as other criteria of the present invention aresatisfied, the ISS can include one or several heterocyclic bases otherthan the principal five base components of naturally-occurring nucleicacids. Preferably, however, the heterocyclic base in the ISS includes,but is not limited to, uracil-5-yl, cytosin-5-yl, adenin-7-yl,adenin-8-yl, guanin-7-yl, guanin-8-yl,4-aminopyrrolo[2.3-d]pyrimidin-5-yl,2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl,2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, where the purines areattached to the sugar moiety of the ISS via the 9-position, thepyrimidines via the 1-position, the pyrrolopyrimidines via the7-position and the pyrazolopyrimidines via the 1-position.

The ISS may comprise at least one modified base. As used herein, theterm “modified base” is synonymous with “base analog,” for example,“modified cytosine” is synonymous with “cytosine analog.” Similarly,“modified” nucleosides or nucleotides are herein defined as beingsynonymous with nucleoside or nucleotide “analogs.” Examples of basemodifications include, but are not limited to, addition of anelectron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the ISS.Preferably, the electron-withdrawing moiety is a halogen. Such modifiedcytosines can include, but are not limited to, azacytosine,5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine,cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine,fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea,iodouracil, 5-nitrocytosine, uracil, and any other pyrimidine analog ormodified pyrimidine. Other examples of base modifications include, butare not limited to, addition of an electron-withdrawing moiety to C-5and/or C-6 of a uracil of the ISS. Preferably, the electron-withdrawingmoiety is a halogen. Such modified uracils can include, but are notlimited to, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and5-iodouracil.

Other examples of base modifications include the addition of one or morethiol groups to the base including, but not limited to, 2-amino-adenine,6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, and4-thio-uracil. Other examples of base modifications include, but are notlimited to, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine and5-hydroxycytosine. See, for example, Kandimalla et al. (2001) Bioorg.Med. Chem. 9:807-813.

The preparation of base-modified nucleosides, and the synthesis ofmodified oligonucleotides using said base-modified nucleosides asprecursors, has been described, for example, in U.S. Pat. Nos.4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosideshave been designed so that they can be incorporated by chemicalsynthesis into either terminal or internal positions of anoligonucleotide. Such base-modified nucleosides, present at eitherterminal or internal positions of an oligonucleotide, can serve as sitesfor attachment of a peptide or other antigen. Nucleosides modified intheir sugar moiety have also been described (including, but not limitedto, e.g., U.S. Pat. Nos. 4,849,513; 5,015,733; 5,118,800; and 5,118,802)and can be used similarly.

In some embodiments, an ISS is less than about any of the followinglengths (in bases or base pairs): 10,000; 5,000; 2500; 2000; 1500; 1250;1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30;25; 20; 15; 14; 13; 12; 11; 10. In some embodiments, an ISS is greaterthan about any of the following lengths (in bases or base pairs): 10;11; 12; 13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200;250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000;50000. Alternately, the ISS can be any of a range of sizes having anupper limit of 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500;300; 250; 200; 175; 150; 125; 100; 75; 60; 50; 40; 30; 25; 20; 15; 14;13; 12; 11; 10 and an independently selected lower limit of 10; 11; 12;13; 14; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250;300; 350; 400; 500; 750; 1000; 2000; 5000; 7500, wherein the lower limitis less than the upper limit. In some embodiments, an ISS is preferablyabout 200 or less bases in length.

Alternatively, ISS may be isolated from microbial species (especiallymycobacteria) using techniques well-known in the art, such as nucleicacid hybridization. Preferably, such isolated ISS will be purified to asubstantially pure state, i.e., to be free of endogenous contaminants,such as lipopolysaccharides. ISS isolated as part of a largerpolynucleotide can be reduced to the desired length by techniques wellknown in the art, such as by endonuclease digestion. Those of ordinaryskill in the art will be familiar with, or can readily ascertain,techniques suitable for isolation, purification and digestion ofpolynucleotides to obtain ISS of potential use in the invention.

Confirmation that a particular oligonucleotide has the properties of anISS useful in the invention can be obtained by evaluating whether theISS affects cytokine secretion as described in infra. Details of invitro techniques useful in making such an evaluation are given in theExamples; those of ordinary skill in the art will also know of, or canreadily ascertain, other methods for measuring cytokine secretion alongthe parameters taught herein.

Antigen that May be Administered with ISS

Any antigen may be co-administered with an ISS and/or used incompositions comprising an ISS and antigen (and preparation of thesecompositions).

In some embodiments, the antigen is an allergen. Examples of recombinantallergens are provided in Table 1. Preparation of many allergens iswell-known in the art, including, but not limited to, preparation ofragweed pollen allergen Antigen E (Amb a I) (Rafnar et al. (1991) J.Biol. Chem. 266:1229-1236), grass allergen Lol p 1 (Tamborini et al.(1997) Eur. J. Biochem. 249:886-894), major dust mite allergens Der pIand Der pI (Chua et al. (1988) J. Exp. Med. 167:175-182; Chua et al.(1990) Int. Arch. Allergy Appl. Immunol. 91:124-129), domestic catallergen Fel d I (Rogers et al. (1993) Mol. Immunol. 30:559-568), whitebirch pollen Bet v1 (Breiteneder et al. (1989) EMBO J. 8:1935-1938),Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al. (2000)Immunology 99:625-629), and protein antigens from other tree pollen(Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl. 204:17-31).As indicated, allergens from trees are known, including allergens frombirch, juniper and Japanese cedar. Preparation of protein antigens fromgrass pollen for in vivo administration has been reported.

In some embodiments, the allergen is a food allergen, including, but notlimited to, peanut allergen, for example Ara h I (Stanley et al. (1996)Adv. Exp. Med. Biol. 409:213-216); walnut allergen, for example, Jug r I(Tueber et al. (1998) J. Allergy Clin. Immunol. 101:807-814); brazil nutallergen, for example, albumin (Pastorello et al. (1998) J. AllergyClin. Immunol. 102:1021-1027; shrISS allergen, for example, Pen a I(Reese et al. (1997) Int. Arch. Allergy Immunol. 113:240-242); eggallergen, for example, ovomucoid (Crooke et al. (1997) J. Immunol.159:2026-2032); milk allergen, for example, bovine β-lactoglobin (Selotal. (1999) Clin. Exp. Allergy 29:1055-1063); fish allergen, for example,parvalbumins (Van Do et al. (1999) Scand. J. Immunol. 50:619-625;Galland et al. (1998) J. Chromatogr. B. Biomed. Sci. Appl. 706:63-71).In some embodiments, the allergen is a latex allergen, including but notlimited to, Hev b 7 (Sowka et al. (1998) Eur. J. Biochem. 255:213-219).Table 1 shows an exemplary list of allergens that may be used.

TABLE 1 RECOMBINANT ALLERGENS Group Allergen Reference ANIMALS:CRUSTACEA ShrISS/lobster tropomyosin Leung et al. (1996) J. AllergyClin. Immunol. 98: 954-961 Pan s I Leung et al. (1998) Mol. Mar. Biol.Biotechnol. 7: 12-20 INSECTS Ant Sol i 2 (venom) Schmidt et al. JAllergy Clin Immunol., 1996, 98: 82-8 Bee Phospholipase A2 (PLA) Mulleret al. J Allergy Clin Immunol, 1995, 96: 395-402 Forster et al. JAllergy Clin Immunol, 1995, 95: 1229-35 Muller et al. Clin Exp Allergy,1997, 27: 915-20 Hyaluronidase (Hya) Soldatova et al. J Allergy ClinImmunol, 1998, 101: 691-8 Cockroach Bla g Bd9OK Helm et al. J AllergyClin Immunol, 1996, 98: 172-180 Bla g 4 (a calycin) Vailes et al. JAllergy Clin Immunol, 1998, 101: 274-280 Glutathione S- Arruda et al. JBiol Chem, 1997, 272: 20907-12 transferase Per a 3 Wu et al. MolImmunol, 1997, 34: 1-8 Dust mite Der p 2 (major allergen) Lynch et al. JAllergy Clin Immunol, 1998, 101: 562-4 Hakkaart et al. Clin Exp Allergy,1998, 28: 169-74 Hakkaart et al. Clin Exp Allergy, 1998, 28: 45-52Hakkaart et al. Int Arch Allergy Immunol, 1998, 115 (2): 150-6 Muelleret al. J Biol Chem, 1997, 272: 26893-8 Der p2 variant Smith et al. JAllergy Clin Immunol, 1998, 101: 423-5 Der f2 Yasue et al. Clin ExpImmunol, 1998, 113: 1-9 Yasue et al. Cell Immunol, 1997, 181: 30-7 Derp10 Asturias et al. Biochim Biophys Acta, 1998, 1397: 27-30 Tyr p 2Eriksson et al. Eur J Biochem, 1998 Hornet Antigen 5 aka Dol m VTomalski et al. Arch Insect Biochem Physiol, 1993, (venom) 22: 303-13Mosquito Aed a I (salivary Xu et al. Int Arch Allergy Immunol, 1998,115: 245-51 apyrase) Yellow jacket antigen 5, hyaluronidase King et al.J Allergy Clin Immunol, 1996, 98: 588-600 and phospholipase (venom)MAMMALS Cat Fel d I Slunt et al. J Allergy Clin Immunol, 1995, 95:1221-8 Hoffmann et al. (1997) J Allergy Clin Immunol 99: 227-32 HedlinCurr Opin Pediatr, 1995, 7: 676-82 Cow Bos d 2 (dander; a Zeiler et al.J Allergy Clin Immunol, 1997, 100: 721-7 lipocalin) Rautiainen et al.Biochem Bioph. Res Comm., 1998, 247: 746-50 β-lactoglobulin (BLG, Chatelet al. Mol Immunol, 1996, 33: 1113-8 major cow milk allergen) Lehrer etal. Crit Rev Food Sci Nutr, 1996, 36: 553-64 Dog Can f I and Can f 2,Konieczny et al. Immunology, 1997, 92: 577-86 salivary lipocalinsSpitzauer et al. J Allergy Clin Immunol, 1994, 93: 614-27 Vrtala et al.J Immunol, 1998, 160: 6137-44 Horse Equ c1 (major allergen, a Gregoireet al. J Biol Chem, 1996, 271: 32951-9 lipocalin) Mouse mouse urinaryprotein Konieczny et al. Immunology, 1997, 92: 577-86 (MUP) OTHERMAMMALIAN ALLERGENS Insulin Ganz et al. J Allergy Clin Immunol, 1990,86: 45-51 Grammer et al. J Lab Clin Med, 1987, 109: 141-6 Gonzalo et al.Allergy, 1998, 53: 106-7 Interferons interferon alpha 2c Detmar et al.Contact Dermatis, 1989, 20: 149-50 MOLLUSCS topomyosin Leung et al. JAllergy Clin Immunol, 1996, 98: 954-61 PLANT ALLERGENS: Barley Hor v 9Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77 Birch pollenallergen, Bet v 4 Twardosz et al. Biochem Bioph. Res Comm., 1997, 23 9:197 rBet v 1 Bet v 2: Pauli et al. J Allergy Clin Immunol, 1996, 97:1100-9 (profilin) van Neerven et al. Clin Exp Allergy, 1998, 28: 423-33Jahn-Schmid et al. Immunotechnology, 1996, 2: 103-13 Breitwieser et al.Biotechniques, 1996, 21: 918-25 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Brazil nut globulin Bartolome et al. AllergolImmunopathol, 1997, 25: 135-44 Cherry Pru a I (major allergen) Scheureret al. Mol Immunol, 1997, 34: 619-29 Corn Zml3 (pollen) Heiss et al.FEBS Lett, 1996, 381: 217-21 Lehrer et al. Int Arch Allergy Immunol,1997, 113: 122-4 Grass Phl p 1, Phl p 2, Phl p 5 Bufe et al. Am J RespirCrit Care Med, 1998, 157: 1269-76 (timothy grass pollen) Vrtala et al. JImmunol Jun. 15, 1998, 160: 6137-44 Niederberger et al. J Allergy ClinImmun., 1998, 101: 258-64 Hol 1 5 velvet grass Schramm et al. Eur JBiochem, 1998, 252: 200-6 pollen Bluegrass allergen Zhang et al. JImmunol, 1993, 151: 791-9 Cyn d 7 Bermuda grass Smith et al. Int ArchAllergy Immunol, 1997, 114: 265-71 Cyn d 12 (a profilin) Asturias et al.Clin Exp Allergy, 1997, 27: 1307-13 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Japanese Cedar Jun a 2 (Juniperus Yokoyama et al.Biochem. Biophys. Res. Commun., ashei) 2000, 275: 195-202 Cry j 1, Cry j2 Kingetsu et al. Immunology, 2000, 99: 625-629 (Cryptomeria japonica)Juniper Jun o 2 (pollen) Tinghino et al. J Allergy Clin Immunol, 1998,101: 772-7 Latex Hev b 7 Sowka et al. Eur J Biochem, 1998, 255: 213-9Fuchs et al. J Allergy Clin Immunol, 1997, 100: 3 56-64 Mercurialis Mera I (profilin) Vallverdu et al. J Allergy Clin Immunol, 1998, 101: 363-70 Mustard Sin a I (seed) Gonzalez de la Pena et al. Biochem Bioph.Res Comm., (Yellow) 1993, 190: 648-53 Oilseed rape Bra r I pollenallergen Smith et al. Int Arch Allergy Immunol, 1997, 114: 265-71 PeanutAra h I Stanley et al. Adv Exp Med Biol, 1996, 409: 213-6 Burks et al. JClin Invest, 1995, 96: 1715-21 Burks et al. Int Arch Allergy Immunol,1995, 107: 248-50 Poa pratensis Poa p9 Parronchi et al. Eur J Immunol,1996, 26: 697-703 Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77Ragweed Amb a I Sun et al. Biotechnology Aug, 1995, 13: 779-86Hirschwehr et al. J Allergy Clin lmmunol, 1998, 101: 196-206 Casale etal. J Allergy Clin Immunol, 1997, 100: 110-21 Rye Lol p I Tamborini etal. Eur J Biochem, 1997, 249: 886-94 Walnut Jug r I Teuber et al. JAllergy Clin Immun., 1998, 101: 807-14 Wheat allergen Fuchs et al. JAllergy Clin Immunol, 1997, 100: 356-64 Donovan et al. Electrophoresis,1993, 14: 917-22 FUNGI: Aspergillus Asp f 1, Asp f 2, Asp f3, Crameri etal. Mycoses, 1998, 41 Suppl 1: 56-60 Asp f 4, rAsp f 6 Hemmann et al.Eur J Immunol, 1998, 28: 1155-60 Banerjee et al. J Allergy Clin Immunol,1997, 99: 821-7 Crameri Int Arch Allergy Immunol, 1998, 115: 99-114Crameri et al. Adv Exp Med Biol, 1996, 409: 111-6 Moser et al. J AllergyClin Immunol, 1994, 93: 1-11 Manganese superoxide Mayer et al. Int ArchAllergy Immunol, 1997, 113: 213-5 dismutase (MNSOD) Blomia allergenCaraballo et al. Adv Exp Med Biol, 1996, 409: 81-3 Penicilliniumallergen Shen et al. Clin Exp Allergy, 1997, 27: 682-90 Psilocybe Psi c2 Horner et al. Int Arch Allergy Immunol, 1995, 107: 298-300

In some embodiments, the antigen is from an infectious agent, includingprotozoan, bacterial, fungal (including unicellular and multicellular),and viral infectious agents. Examples of suitable viral antigens aredescribed herein and are known in the art. Bacteria include Hemophilusinfluenza, Mycobacterium tuberculosis and Bordetella pertussis.Protozoan infectious agents include malarial plasmodia, Leishmaniaspecies, Trypanosoma species and Schistosoma species. Fungi includeCandida albicans.

In some embodiments, the antigen is a viral antigen. Viral polypeptideantigens include, but are not limited to, HIV proteins such as HIV gagproteins (including, but not limited to, membrane anchoring (MA)protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIVpolymerase, influenza virus matrix (M) protein and influenza virusnucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg),hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitisB DNA polymerase, hepatitis C antigens, and the like. Referencesdiscussing influenza vaccination include Scherle and Gerhard (1988)Proc. Natl. Acad. Sci. USA 85:4446-4450; Scherle and Gerhard (1986) J.Exp. Med. 164:1114-1128; Granoff et al. (1993) Vaccine 11:S46-51;Kodihalli et al. (1997) J. Virol. 71:3391-3396; Ahmeida et al. (1993)Vaccine 11:1302-1309; Chen et al. (1999) Vaccine 17:653-659; Govorkovaand Smirnov (1997) Acta Virol. (1997) 41:251-257; Koide et al. (1995)Vaccine 13:3-5; Mbawuike et al. (1994) Vaccine 12:1340-1348; Tamura etal. (1994) Vaccine 12:310-316; Tamura et al. (1992) Eur. J. Immunol.22:477-481; Hirabayashi et al. (1990) Vaccine 8:595-599. Other examplesof antigen polypeptides are group- or sub-group specific antigens, whichare known for a number of infectious agents, including, but not limitedto, adenovirus, herpes simplex virus, papilloma virus, respiratorysyncytial virus and poxviruses.

Many antigenic peptides and proteins are known, and available in theart; others can be identified using conventional techniques. Forimmunization against tumor formation or treatment of existing tumors,immunomodulatory peptides can include tumor cells (live or irradiated),tumor cell extracts, or protein subunits of tumor antigens such asHer-2/neu, Mart1, carcinoembryonic antigen (CEA), gangliosides, humanmilk fat globule (HMFG), mucin (MUC1), MAGE antigens, BAGE antigens,GAGE antigens, gp100, prostate specific antigen (PSA), and tyrosinase.Vaccines for immuno-based contraception can be formed by including spermproteins administered with ISS. Lea et al. (1996) Biochim. Biophys. Acta1307:263.

Attenuated and inactivated viruses are suitable for use herein as theantigen. Preparation of these Viruses is Well-Known in the Art and Manyare Commercially Available (See, e.g., Physicians' Desk Reference (1998)52nd edition, Medical Economics Company, Inc.). For example, polio virusis available as IPOL® (Pasteur Merieux Connaught) and ORIMUNE® (LederleLaboratories), hepatitis A virus as VAQTA® (Merck), measles virus asATTENUVAX® (Merck), mumps virus as MUMPSVAX® (Merck) and rubella virusas MERUVAX®II (Merck). Additionally, attenuated and inactivated virusessuch as HIV-1, HIV-2, herpes simplex virus, hepatitis B virus,rotavirus, human and non-human papillomavirus and slow brain viruses canprovide peptide antigens. In some embodiments, the antigen comprises aviral vector, such as vaccinia, adenovirus, and canary pox.

Antigens may be isolated from their source using purification techniquesknown in the art or, more conveniently, may be produced usingrecombinant methods. Antigenic peptides can include purified nativepeptides, synthetic peptides, recombinant proteins, crude proteinextracts, attenuated or inactivated viruses, cells, micro-organisms, orfragments of such peptides. Immunomodulatory peptides can be native orsynthesized chemically or enzymatically. Any method of chemicalsynthesis known in the art is suitable. Solution phase peptide synthesiscan be used to construct peptides of moderate size or, for the chemicalconstruction of peptides, solid phase synthesis can be employed.Atherton et al. (1981) Hoppe Seylers Z. Physiol. Chem. 362:833-839.Proteolytic enzymes can also be utilized to couple amino acids toproduce peptides. Kullmann (1987) Enzymatic Peptide Synthesis, CRCPress, Inc. Alternatively, the peptide can be obtained by using thebiochemical machinery of a cell, or by isolation from a biologicalsource. Recombinant DNA techniques can be employed for the production ofpeptides. Hames et al. (1987) Transcription and Translation: A PracticalApproach, IRL Press. Peptides can also be isolated using standardtechniques such as affinity chromatography.

Preferably the antigens are peptides, lipids (e.g., sterols excludingcholesterol, fatty acids, and phospholipids), polysaccharides such asthose used in H. influenza vaccines, gangliosides and glycoproteins.These can be obtained through several methods known in the art,including isolation and synthesis using chemical and enzymatic methods.In certain cases, such as for many sterols, fatty acids andphospholipids, the antigenic portions of the molecules are commerciallyavailable.

Examples of viral antigens useful in the subject compositions andmethods using the compositions include, but are not limited to, HIVantigens. Such antigens include, but are not limited to, those antigensderived from HIV envelope glycoproteins including, but not limited to,gp160, gp120 and gp41. Numerous sequences for HIV genes and antigens areknown. For example, the Los Alamos National Laboratory HIV SequenceDatabase collects, curates and annotates HIV nucleotide and amino acidsequences. This database is accessible via the internet and in a yearlypublication, see Human Retroviruses and AIDS Compendium (for example,2000 edition).

Antigens derived from infectious agents may be obtained using methodsknown in the art, for example, from native viral or bacterial extracts,from cells infected with the infectious agent, from purifiedpolypeptides, from recombinantly produced polypeptides and/or assynthetic peptides.

ISS-Antigen

When used with antigen, ISS may be administered with antigen in a numberof ways. In some embodiments, an ISS and antigen may be administeredspatially proximate with respect to each other, or as an admixture(i.e., in solution). As described below, spatial proximation can beaccomplished in a number of ways, including conjugation (linkage),encapsidation, via affixation to a platform or adsorption onto asurface. Generally, and most preferably, an ISS and antigen areproximately associated at a distance effective to enhance the immuneresponse generated compared to the administration of the ISS and theantigen as an admixture.

In some embodiments, the ISS is conjugated with the antigen. The ISSportion can be coupled with the antigen portion of a conjugate in avariety of ways, including covalent and/or non-covalent interactions.

The link between the portions can be made at the 3′ or 5′ end of theISS, or at a suitably modified base at an internal position in the ISS.If the antigen is a peptide and contains a suitable reactive group(e.g., an N-hydroxysuccinimide ester) it can be reacted directly withthe N⁴ amino group of cytosine residues. Depending on the number andlocation of cytosine residues in the ISS, specific coupling at one ormore residues can be achieved.

Alternatively, modified oligonucleosides, such as are known in the art,can be incorporated at either terminus, or at internal positions in theISS. These can contain blocked functional groups which, when deblocked,are reactive with a variety of functional groups which can be presenton, or attached to, the antigen of interest.

Where the antigen is a peptide or polypeptide, this portion of theconjugate can be attached to the 3′-end of the ISS through solid supportchemistry. For example, the ISS portion can be added to a polypeptideportion that has been pre-synthesized on a support. Haralambidis et al.(1990a) Nucleic Acids Res. 18:493-499; and Haralambidis et al. (1990b)Nucleic Acids Res. 18:501-505. Alternatively, the ISS can be synthesizedsuch that it is connected to a solid support through a cleavable linkerextending from the 3′-end. Upon chemical cleavage of the ISS from thesupport, a terminal thiol group is left at the 3′-end of theoligonucleotide (Zuckermann et al. (1987) Nucleic Acids Res.15:5305-5321; and Corey et al. (1987) Science 238:1401-1403) or aterminal amino group is left at the 3′-end of the oligonucleotide(Nelson et al. (1989) Nucleic Acids Res. 17:1781-1794). Conjugation ofthe amino-modified ISS to amino groups of the peptide can be performedas described in Benoit et al. (1987) Neuromethods 6:43-72. Conjugationof the thiol-modified ISS to carboxyl groups of the peptide can beperformed as described in Sinah et al. (1991) Oligonucleotide Analogues:A Practical Approach, IRL Press. Coupling of an oligonucleotide carryingan appended maleimide to the thiol side chain of a cysteine residue of apeptide has also been described. Tung et al. (1991) Bioconjug. Chem.2:464-465.

The peptide or polypeptide portion of the conjugate can be attached tothe 5′-end of the ISS through an amine, thiol, or carboxyl group thathas been incorporated into the oligonucleotide during its synthesis.Preferably, while the oligonucleotide is fixed to the solid support, alinking group comprising a protected amine, thiol, or carboxyl at oneend, and a phosphoramidite at the other, is covalently attached to the5′-hydroxyl. Subsequent to deprotection, the amine, thiol, and carboxylfunctionalities can be used to covalently attach the oligonucleotide toa peptide. Benoit et al. (1987); and Sinah et al. (1991).

An ISS-antigen conjugate can also be formed through non-covalentinteractions, such as ionic bonds, hydrophobic interactions, hydrogenbonds and/or van der Waals attractions.

Non-covalently linked conjugates can include a non-covalent interactionsuch as a biotin-streptavidin complex. A biotinyl group can be attached,for example, to a modified base of an ISS. Roget et al. (1989) NucleicAcids Res. 17:7643-7651. Incorporation of a streptavidin moiety into thepeptide portion allows formation of a non-covalently bound complex ofthe streptavidin conjugated peptide and the biotinylatedoligonucleotide.

Non-covalent associations can also occur through ionic interactionsinvolving an ISS and residues within the antigen, such as charged aminoacids, or through the use of a linker portion comprising chargedresidues that can interact with both the oligonucleotide and theantigen. For example, non-covalent conjugation can occur between agenerally negatively-charged ISS and positively-charged amino acidresidues of a peptide, e.g., polylysine, polyarginine and polyhistidineresidues.

Non-covalent conjugation between ISS and antigens can occur through DNAbinding motifs of molecules that interact with DNA as their naturalligands. For example, such DNA binding motifs can be found intranscription factors and anti-DNA antibodies.

The linkage of the ISS to a lipid can be formed using standard methods.These methods include, but are not limited to, the synthesis ofoligonucleotide-phospholipid conjugates (Yanagawa et al. (1988) NucleicAcids Symp. Ser. 19:189-192), oligonucleotide-fatty acid conjugates(Grabarek et al. (1990) Anal. Biochem. 185:131-135; and Staros et al.(1986) Anal. Biochem. 156:220-222), and oligonucleotide-sterolconjugates. Boujrad et al. (1993) Proc. Natl. Acad. Sci. USA90:5728-5731.

The linkage of the oligonucleotide to an oligosaccharide can be formedusing standard known methods. These methods include, but are not limitedto, the synthesis of oligonucleotide-oligosaccharide conjugates, whereinthe oligosaccharide is a moiety of an immunoglobulin. O'Shannessy et al.(1985) J. Applied Biochem. 7:347-355.

The linkage of a circular ISS to a peptide or antigen can be formed inseveral ways. Where the circular ISS is synthesized using recombinant orchemical methods, a modified nucleoside is suitable. Ruth (1991) inOligonucleotides and Analogues: A Practical Approach, IRL Press.Standard linking technology can then be used to connect the circular ISSto the antigen or other peptide. Goodchild (1990) Bioconjug. Chem.1:165. Where the circular ISS is isolated, or synthesized usingrecombinant or chemical methods, the linkage can be formed by chemicallyactivating, or photoactivating, a reactive group (e.g. carbene, radical)that has been incorporated into the antigen or other peptide.

Additional methods for the attachment of peptides and other molecules tooligonucleotides can be found in U.S. Pat. No. 5,391,723; Kessler (1992)“Nonradioactive labeling methods for nucleic acids” in Kricka (ed.)Nonisotopic DNA Probe Techniques, Academic Press; and Geoghegan et al.(1992) Bioconjug. Chem. 3:138-146.

An ISS may be proximately associated with an antigen(s) in other ways.In some embodiments, an ISS and antigen are proximately associated byencapsulation. In other embodiments, an ISS and antigen are proximatelyassociated by linkage to a platform molecule. A “platform molecule”(also termed “platform”) is a molecule containing sites which allow forattachment of the ISS and antigen(s). In other embodiments, an ISS andantigen are proximately associated by adsorption onto a surface,preferably a carrier particle.

In some embodiments, the methods of the invention employ anencapsulating agent that can maintain the proximate association of theISS and first antigen until the complex is available to the target (orcompositions comprising such encapsulating agents). Preferably, thecomposition comprising ISS, antigen and encapsulating agent is in theform of adjuvant oil-in-water emulsions, microparticles and/orliposomes. More preferably, adjuvant oil-in-water emulsions,microparticles and/or liposomes encapsulating an ISS-immunomodulatorymolecule are in the form of particles from about 0.04 μm to about 100 μmin size, preferably any of the following ranges: from about 0.1 μm toabout 20 μm; from about 0.15 μm to about 10 μm; from about 0.05 μm toabout 1.00 μm; from about 0.05 μm to about 0.5 μm.

Colloidal dispersion systems, such as microspheres, beads,macromolecular complexes, nanocapsules and lipid-based system, such asoil-in-water emulsions, micelles, mixed micelles and liposomes canprovide effective encapsulation of ISS-containing compositions.

The encapsulation composition further comprises any of a wide variety ofcomponents. These include, but are not limited to, alum, lipids,phospholipids, lipid membrane structures (LMS), polyethylene glycol(PEG) and other polymers, such as polypeptides, glycopeptides, andpolysaccharides.

Polypeptides suitable for encapsulation components include any known inthe art and include, but are not limited to, fatty acid bindingproteins. Modified polypeptides contain any of a variety ofmodifications, including, but not limited to glycosylation,phosphorylation, myristylation, sulfation and hydroxylation. As usedherein, a suitable polypeptide is one that will protect anISS-containing composition to preserve the immunomodulatory activitythereof. Examples of binding proteins include, but are not limited to,albumins such as bovine serum albumin (BSA) and pea albumin.

Other suitable polymers can be any known in the art of pharmaceuticalsand include, but are not limited to, naturally-occurring polymers suchas dextrans, hydroxyethyl starch, and polysaccharides, and syntheticpolymers. Examples of naturally occurring polymers include proteins,glycopeptides, polysaccharides, dextran and lipids. The additionalpolymer can be a synthetic polymer. Examples of synthetic polymers whichare suitable for use in the present invention include, but are notlimited to, polyalkyl glycols (PAG) such as PEG, polyoxyethylatedpolyols (POP), such as polyoxyethylated glycerol (POG), polytrimethyleneglycol (PTG) polypropylene glycol (PPG), polyhydroxyethyl methacrylate,polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline,polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids,polyurethane and polyphosphazene. The synthetic polymers can also belinear or branched, substituted or unsubstituted, homopolymeric,co-polymers, or block co-polymers of two or more different syntheticmonomers.

The PEGs for use in encapsulation compositions of the present inventionare either purchased from chemical suppliers or synthesized usingtechniques known to those of skill in the art.

The term “LMS”, as used herein, means lamellar lipid particles whereinpolar head groups of a polar lipid are arranged to face an aqueous phaseof an interface to form membrane structures. Examples of the LMSsinclude liposomes, micelles, cochleates (i.e., generally cylindricalliposomes), microemulsions, unilamellar vesicles, multilamellarvesicles, and the like.

A preferred colloidal dispersion system of this invention is a liposome.In mice immunized with a liposome-encapsulated antigen, liposomesappeared to enhance a Th1-type immune response to the antigen. Aramakiet al. (1995) Vaccine 13:1809-1814. As used herein, a “liposome” or“lipid vesicle” is a small vesicle bounded by at least one and possiblymore than one bilayer lipid membrane. Liposomes are made artificiallyfrom phospholipids, glycolipids, lipids, steroids such as cholesterol,related molecules, or a combination thereof by any technique known inthe art, including but not limited to sonication, extrusion, or removalof detergent from lipid-detergent complexes. A liposome can alsooptionally comprise additional components, such as a tissue targetingcomponent. It is understood that a “lipid membrane” or “lipid bilayer”need not consist exclusively of lipids, but can additionally contain anysuitable other components, including, but not limited to, cholesteroland other steroids, lipid-soluble chemicals, proteins of any length, andother amphipathic molecules, providing the general structure of themembrane is a sheet of two hydrophilic surfaces sandwiching ahydrophobic core. For a general discussion of membrane structure, seeThe Encyclopedia of Molecular Biology by J. Kendrew (1994). For suitablelipids see e.g., Lasic (1993) “Liposomes: from Physics to Applications”Elsevier, Amsterdam.

Processes for preparing liposomes containing ISS-containing compositionsare known in the art. The lipid vesicles can be prepared by any suitabletechnique known in the art. Methods include, but are not limited to,microencapsulation, microfluidization, LLC method, ethanol injection,freon injection, the “bubble” method, detergent dialysis, hydration,sonication, and reverse-phase evaporation. Reviewed in Watwe et al.(1995) Curr. Sci. 68:715-724. Techniques may be combined in order toprovide vesicles with the most desirable attributes.

The invention encompasses use of LMSs containing tissue or cellulartargeting components. Such targeting components are components of a LMSthat enhance its accumulation at certain tissue or cellular sites inpreference to other tissue or cellular sites when administered to anintact animal, organ, or cell culture. A targeting component isgenerally accessible from outside the liposome, and is thereforepreferably either bound to the outer surface or inserted into the outerlipid bilayer. A targeting component can be inter alia a peptide, aregion of a larger peptide, an antibody specific for a cell surfacemolecule or marker, or antigen binding fragment thereof, a nucleic acid,a carbohydrate, a region of a complex carbohydrate, a special lipid, ora small molecule such as a drug, hormone, or hapten, attached to any ofthe aforementioned molecules. Antibodies with specificity toward celltype-specific cell surface markers are known in the art and are readilyprepared by methods known in the art.

The LMSs can be targeted to any cell type toward which a therapeutictreatment is to be directed, e.g., a cell type which can modulate and/orparticipate in an immune response. Such target cells and organs include,but are not limited to, APCs, such as macrophages, dendritic cells andlymphocytes, lymphatic structures, such as lymph nodes and the spleen,and nonlymphatic structures, particularly those in which dendritic cellsare found.

The LMS compositions of the present invention can additionally comprisesurfactants. Surfactants can be cationic, anionic, amphiphilic, ornonionic. One class of surfactants that can be used is nonionicsurfactants; particularly preferred are those that are water soluble.

In embodiments in which an ISS and antigen are proximately associated bylinkage to a platform molecule, the platform may be proteinaceous ornon-proteinaceous (i.e., organic). Examples of proteinaceous platformsinclude, but are not limited to, albumin, gammaglobulin, immunoglobulin(IgG) and ovalbumin. Borel et al. (1990) Immunol. Methods 126:159-168;Dumas et al. (1995) Arch. Dematol. Res. 287:123-128; Borel et al. (1995)Int. Arch. Allergy Immunol. 107:264-267; Borel et al. (1996) Ann. N.Y.Acad. Sci. 778:80-87. A platform is multi-valent (i.e., contains morethan one binding, or linking, site) to accommodate binding to both anISS and antigen. Accordingly, a platform may contain 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more binding or linking sites Other examples of polymericplatforms are dextran, polyacrylamide, ficoll, carboxymethylcellulose,polyvinyl alcohol, and poly D-glutamic acid/D-lysine.

The principles of using platform molecules are well understood in theart. Generally, a platform contains, or is derivatized to contain,appropriate binding sites for ISS and antigen. In addition, oralternatively, ISS and/or antigen is derivatized to provide appropriatelinkage groups. For example, a simple platform is a bi-functional linker(i.e., has two binding sites), such as a peptide. Further examples arediscussed below.

Platform molecules may be biologically stabilized, i.e., they exhibit anin vivo excretion half-life often of hours to days to months to confertherapeutic efficacy, and are preferably composed of a synthetic singlechain of defined composition. They generally have a molecular weight inthe range of about 200 to about 1,000,000, preferably any of thefollowing ranges: from about 200 to about 500,000; from about 200 toabout 200,000; from about 200 to about 50,000 (or less, such as 30,000).Examples of valency platform molecules are polymers (or are comprised ofpolymers) such as polyethylene glycol (PEG; preferably having amolecular weight of about 200 to about 8000), poly-D-lysine, polyvinylalcohol, polyvinylpyrrolidone, D-glutamic acid and D-lysine (in a ratioof 3:2). Other molecules that may be used are albumin and IgG.

Other platform molecules suitable for use within the present inventionare the chemically-defined, non-polymeric valency platform moleculesdisclosed in U.S. Pat. No. 5,552,391. Other homogeneouschemically-defined valency platform molecules suitable for use withinthe present invention are derivatized 2,2′-ethylenedioxydiethylamine(EDDA) and triethylene glycol (TEG).

Additional suitable valency platform molecules include, but are notlimited to, tetraminobenzene, heptaminobetacyclodextrin,tetraminopentaerythritol, 1,4,8,11-tetraazacyclotetradecane (Cyclam) and1,4,7,10-tetraazacyclododecane (Cyclen).

In general, these platforms are made by standard chemical synthesistechniques. PEG must be derivatized and made multivalent, which isaccomplished using standard techniques. Some substances suitable forconjugate synthesis, such as PEG, albumin, and IgG are availablecommercially.

Conjugation of an ISS and antigen to a platform molecule may be effectedin any number of ways, typically involving one or more crosslinkingagents and functional groups on the antigen and ISS platform andplatform molecule. Platforms and ISS and antigen must have appropriatelinking groups. Linking groups are added to platforms using standardsynthetic chemistry techniques. Linking groups may be added topolypeptide antigens and ISS using either standard solid phase synthetictechniques or recombinant techniques. Recombinant approaches may requirepost-translational modification in order to attach a linker, and suchmethods are known in the art.

As an example, polypeptides contain amino acid side chain moietiescontaining functional groups such as amino, carboxyl or sulfhydrylgroups that serve as sites for coupling the polypeptide to the platform.Residues that have such functional groups may be added to thepolypeptide if the polypeptide does not already contain these groups.Such residues may be incorporated by solid phase synthesis techniques orrecombinant techniques, both of which are well known in the peptidesynthesis arts. When the polypeptide has a carbohydrate side chain(s)(or if the antigen is a carbohydrate), functional amino, sulfhydryland/or aldehyde groups may be incorporated therein by conventionalchemistry. For instance, primary amino groups may be incorporated byreaction of the oxidized sugar with ethylenediamine in the presence ofsodium cyanoborohydride, sulfhydryls may be introduced by reaction ofcysteamine dihydrochloride followed by reduction with a standarddisulfide reducing agent, while aldehyde groups may be generatedfollowing periodate oxidation. In a similar fashion, the platformmolecule may also be derivatized to contain functional groups if it doesnot already possess appropriate functional groups.

Hydrophilic linkers of variable lengths are useful for connecting ISSand antigen to platform molecules. Suitable linkers include linearoligomers or polymers of ethylene glycol. Such linkers include linkerswith the formula R¹S(CH₂CH₂O)_(n)CH₂CH₂O(CH₂)_(m)CO₂R² wherein n=0-200,m=1 or 2, R¹=H or a protecting group such as trityl, R²=H or alkyl oraryl, e.g., 4-nitrophenyl ester. These linkers are useful in connectinga molecule containing a thiol reactive group such as haloaceyl,maleiamide, etc., via a thioether to a second molecule which contains anamino group via an amide bond. These linkers are flexible with regard tothe order of attachment, i.e., the thioether can be formed first orlast.

In embodiments in which an ISS and antigen are proximately associated byadsorption onto a surface, the surface may be in the form of a carrierparticle (for example, a nanoparticle) made with either an inorganic ororganic core. Examples of such nanoparticles include, but are notlimited to, nanocrystalline particles, nanoparticles made by thepolymerization of alkylcyanoacrylates and nanoparticles made by thepolymerization of methylidene malonate. Additional surfaces to which anISS and antigen may be adsorbed include, but are not limited to,activated carbon particles and protein-ceramic nanoplates. Otherexamples of carrier particles are provided herein.

Adsorption of polynucleotides and polypeptides to a surface for thepurpose of delivery of the adsorbed molecules to cells is well known inthe art. See, for example, Douglas et al. (1987) Crit. Rev. Ther. Drug.Carrier Syst. 3:233-261; Hagiwara et al. (1987) In Vivo 1:241-252;Bousquet et al. (1999) Pharm. Res. 16:141-147; and Kossovsky et al.,U.S. Pat. No. 5,460,831. Preferably, the material comprising theadsorbent surface is biodegradable. Adsorption of an ISS and/or antigento a surface may occur through non-covalent interactions, includingionic and/or hydrophobic interactions.

In general, characteristics of carriers such as nanoparticles, such assurface charge, particle size and molecular weight, depend uponpolymerization conditions, monomer concentration and the presence ofstabilizers during the polymerization process (Douglas et al., 1987).The surface of carrier particles may be modified, for example, with asurface coating, to allow or enhance adsorption of the ISS and/orantigen. Carrier particles with adsorbed ISS and/or antigen may befurther coated with other substances. The addition of such othersubstances may, for example, prolong the half-life of the particles onceadministered to the subject and/or may target the particles to aspecific cell type or tissue, as described herein.

Nanocrystalline surfaces to which an ISS and antigen may be adsorbedhave been described (see, for example, U.S. Pat. No. 5,460,831).Nanocrystalline core particles (with diameters of 1 μm or less) arecoated with a surface energy modifying layer that promotes adsorption ofpolypeptides, polynucleotides and/or other pharmaceutical agents. Asdescribed in U.S. Pat. No. 5,460,831, for example, a core particle iscoated with a surface that promotes adsorption of an oligonucleotide andis subsequently coated with an antigen preparation, for example, in theform of a lipid-antigen mixture. Such nanoparticles are self-assemblingcomplexes of nanometer sized particles, typically on the order of 0.1μm, that carry an inner layer of ISS and an outer layer of antigen.

Another adsorbent surface are nanoparticles made by the polymerizationof alkylcyanoacrylates. Alkylcyanoacrylates can be polymerized inacidified aqueous media by a process of anionic polymerization.Depending on the polymerization conditions, the small particles tend tohave sizes in the range of 20 to 3000 nm, and it is possible to makenanoparticles specific surface characteristics and with specific surfacecharges (Douglas et al., 1987). For example, oligonucleotides may beadsorbed to polyisobutyl- and polyisohexlcyanoacrylate nanoparticles inthe presence of hydrophobic cations such as tetraphenylphosphoniumchloride or quaternary ammonium salts, such as cetyltrimethyl ammoniumbromide. Oligonucleotide adsorption on these nanoparticles appears to bemediated by the formation of ion pairs between negatively chargedphosphate groups of the nucleic acid chain and the hydrophobic cations.See, for example, Lambert et al. (1998) Biochimie 80:969-976, Chavany etal. (1994) Pharm. Res. 11:1370-1378; Chavany et al. (1992) Pharm. Res.9:441-449. Polypeptides may also be adsorbed to polyalkylcyanoacrylatenanoparticles. See, for example, Douglas et al., 1987; Schroeder et al.(1998) Peptides 19:777-780.

Another adsorbent surface is nanoparticles made by the polymerization ofmethylidene malonate. For example, as described in Bousquet et al.,1999, polypeptides adsorbed to poly(methylidene malonate 2.1.2)nanoparticles appear to do so initially through electrostatic forcesfollowed by stabilization through hydrophobic forces.

ISS/MC Complexes

ISSs may be administered in the form of ISS/microcarrier (ISS/MC)complexes. Accordingly, the invention provides compositions comprisingISS/MC complexes.

Microcarriers useful in the invention are less than about 150, 120 or100 μm in size, more commonly less than about 50-60 μm in size,preferably less than about 10 μm in size, and are insoluble in purewater. Microcarriers used in the invention are preferably biodegradable,although nonbiodegradable microcarriers are acceptable. Microcarriersare commonly solid phase, such as “beads” or other particles, althoughliquid phase microcarriers such as oil in water emulsions comprising abiodegradable polymers or oils are also contemplated. A wide variety ofbiodegradable and nonbiodegradable materials acceptable for use asmicrocarriers are known in the art.

Microcarriers for use in the compositions or methods of the inventionare generally less than about 10 μm in size (e.g., have an averagediameter of less than about 10 μm, or at least about 97% of theparticles pass through a 10 μm screen filter), and include nanocarriers(i.e., carriers of less than about 1 μm size). Preferably, microcarriersare selected having sizes within an upper limit of about 9, 7, 5, 2, or1 μm or 900, 800, 700, 600, 500, 400, 300, 250, 200, or 100 nm and anindependently selected lower limit of about 4, 2, or 1 μm or about 800,600, 500, 400, 300, 250, 200, 150, 100, 50, 25, or 10 nm, where thelower limit is less than the upper limit. In some embodiments, themicrocarriers have a size of about 1.0-1.5 μm, about 1.0-2.0 μm or about0.9-1.6 μm. In certain preferred embodiments, the microcarriers have asize of about 10 nm to about 5 μm or about 25 nm to about 4.5 μm, about1 μm, about 1.2 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.8μm, about 2.0 μm, about 2.5 μm or about 4.5 μm. When the microcarriersare nanocarriers, preferred embodiments include nanocarriers of about 25to about 300 nm, 50 to about 200 nm, about 50 nm or about 200 nm.

Solid phase biodegradable microcarriers may be manufactured frombiodegradable polymers including, but not limited to: biodegradablepolyesters, such as poly(lactic acid), poly(glycolic acid), andcopolymers (including block copolymers) thereof, as well as blockcopolymers of poly(lactic acid) and poly(ethylene glycol);polyorthoesters such as polymers based on3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU);polyanhydrides such as poly(anhydride) polymers based on relativelyhydrophilic monomers such as sebacic acid; polyanhydride imides, such aspolyanhydride polymers based on sebacic acid-derived monomersincorporating amino acids (i.e., linked to sebacic acid by imide bondsthrough the amino-terminal nitrogen) such as glycine or alanine;polyanhydride esters; polyphosphazenes, especially poly(phosphazenes)which contain hydrolysis-sensitive ester groups which can catalyzedegradation of the polymer backbone through generation of carboxylicacid groups (Schacht et al., (1996) Biotechnol. Bioeng. 1996:102); andpolyamides such as poly(lactic acid-co-lysine).

A wide variety of nonbiodegradable materials suitable for manufacturingmicrocarriers are also known, including, but not limited to polystyrene,polypropylene, polyethylene, silica, ceramic, polyacrylamide, dextran,hydroxyapatite, latex, gold, and ferromagnetic or paramagneticmaterials. Certain embodiments exclude gold, latex, and/or magneticbeads. In certain embodiments, the microcarriers may be made of a firstmaterial (e.g., a magnetic material) encapsulated with a second material(e.g., polystyrene).

Solid phase microspheres are prepared using techniques known in the art.For example, they can be prepared by emulsion-solventextraction/evaporation technique. Generally, in this technique,biodegradable polymers such as polyanhydrates,poly(alkyl-α-cyanoacrylates) and poly(α-hydroxy esters), for example,poly(lactic acid), poly(glycolic acid), poly(D,L-lactic-co-glycolicacid) and poly(caprolactone), are dissolved in a suitable organicsolvent, such as methylene chloride, to constitute the dispersed phase(DP) of emulsion. DP is emulsified by high-speed homogenization intoexcess volume of aqueous continuous phase (CP) that contains a dissolvedsurfactant, for example, polyvinylalcohol (PVA) or polyvinylpirrolidone(PVP). Surfactant in CP is to ensure the formation of discrete andsuitably-sized emulsion droplet. The organic solvent is then extractedinto the CP and subsequently evaporated by raising the systemtemperature. The solid microparticles are then separated bycentrifugation or filtration, and dried, for example, by lyophilizationor application of vacuum, before storing at 4° C.

Physico-chemical characteristics such as mean size, size distributionand surface charge of dried microspheres may be determined. Sizecharacteristics are determined, for example, by dynamic light scatteringtechnique and the surface charge was determined by measuring the zetapotential.

Liquid phase microcarriers include liposomes, micelles, oil droplets andother lipid or oil-based particles which incorporate biodegradablepolymers or oils. In certain embodiments, the biodegradable polymer is asurfactant. In other embodiments, the liquid phase microcarriers arebiodegradable due to the inclusion of a biodegradable oil such assqualene or a vegetable oil. One preferred liquid phase microcarrier isoil droplets within an oil-in-water emulsion. Preferably, oil-in-wateremulsions used as microcarriers comprise biodegradable substituents suchas squalene.

ISS/MC complexes comprise an ISS bound to the surface of a microcarrier(i.e., the ISS is not encapsulated in the MC), and preferably comprisemultiple molecules of ISS bound to each microcarrier. In certainembodiments, a mixture of different ISSs may be complexed with amicrocarrier, such that the microcarrier is bound to more than one ISSspecies. The bond between the ISS and MC may be covalent ornon-covalent. As will be understood by one of skill in the art, the ISSmay be modified or derivatized and the composition of the microcarriermay be selected and/or modified to accommodate the desired type ofbinding desired for ISS/MC complex formation.

Covalently bonded ISS/MC complexes may be linked using any covalentcrosslinking technology known in the art. Typically, the ISS portionwill be modified, either to incorporate an additional moiety (e.g., afree amine, carboxyl or sulfhydryl group) or incorporate modified (e.g.,phosphorothioate) nucleotide bases to provide a site at which the ISSportion may be linked to the microcarrier. The link between the ISS andMC portions of the complex can be made at the 3′ or 5′ end of the ISS,or at a suitably modified base at an internal position in the ISS. Themicrocarrier is generally also modified to incorporate moieties throughwhich a covalent link may be formed, although functional groups normallypresent on the microcarrier may also be utilized. The ISS/MC is formedby incubating the ISS with a microcarrier under conditions which permitthe formation of a covalent complex (e.g., in the presence of acrosslinking agent or by use of an activated microcarrier comprising anactivated moiety which will form a covalent bond with the ISS).

A wide variety of crosslinking technologies are known in the art, andinclude crosslinkers reactive with amino, carboxyl and sulfhydrylgroups. As will be apparent to one of skill in the art, the selection ofa crosslinking agent and crosslinking protocol will depend on theconfiguration of the ISS and the microcarrier as well as the desiredfinal configuration of the ISS/MC complex. The crosslinker may be eitherhomobifunctional or heterobifunctional. When a homobifunctionalcrosslinker is used, the crosslinker exploits the same moiety on the ISSand MC (e.g., an aldehyde crosslinker may be used to covalently link anISS and MC where both the ISS and MC comprise one or more free amines).Heterobifunctional crosslinkers utilize different moieties on the ISSand MC, (e.g., a maleimido-N-hydroxysuccinimide ester may be used tocovalently link a free sulfhydryl on the ISS and a free amine on theMC), and are preferred to minimize formation of inter-microcarrierbonds. In most cases, it is preferable to crosslink through a firstcrosslinking moiety on the microcarrier and a second crosslinking moietyon the ISS, where the second crosslinking moiety is not present on themicrocarrier. One preferred method of producing the ISS/MC complex is by‘activating’ the microcarrier by incubating with a heterobifunctionalcrosslinking agent, then forming the ISS/MC complex by incubating theISS and activated MC under conditions appropriate for reaction. Thecrosslinker may incorporate a “spacer” arm between the reactivemoieties, or the two reactive moieties in the crosslinker may bedirectly linked.

In one preferred embodiment, the ISS portion comprises at least one freesulfhydryl (e.g., provided by a 5′-thiol modified base or linker) forcrosslinking to the microcarrier, while the microcarrier comprises freeamine groups. A heterobifunctional crosslinker reactive with these twogroups (e.g., a crosslinker comprising a maleimide group and aNHS-ester), such as succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate is used to activate theMC, then covalently crosslink the ISS to form the ISS/MC complex.

Non-covalent ISS/MC complexes may be linked by any non-covalent bindingor interaction, including ionic (electrostatic) bonds, hydrophobicinteractions, hydrogen bonds, van der Waals attractions, or acombination of two or more different interactions, as is normally thecase when a binding pair is to link the ISS and MC.

Preferred non-covalent ISS/MC complexes are typically complexed byhydrophobic or electrostatic (ionic) interactions, or a combinationthereof, (e.g., through base pairing between an ISS and a polynucleotidebound to an MC use of a binding pair). Due to the hydrophilic nature ofthe backbone of polynucleotides, ISS/MC complexes which rely onhydrophobic interactions to form the complex generally requiremodification of the ISS portion of the complex to incorporate a highlyhydrophobic moiety. Preferably, the hydrophobic moiety is biocompatible,nonimmunogenic, and is naturally occurring in the individual for whomthe composition is intended (e.g., is found in mammals, particularlyhumans). Examples of preferred hydrophobic moieties include lipids,steroids, sterols such as cholesterol, and terpenes. The method oflinking the hydrophobic moiety to the ISS will, of course, depend on theconfiguration of the ISS and the identity of the hydrophobic moiety. Thehydrophobic moiety may be added at any convenient site in the ISS,preferably at either the 5′ or 3′ end; in the case of addition of acholesterol moiety to an ISS, the cholesterol moiety is preferably addedto the 5′ end of the ISS, using conventional chemical reactions (see,for example, Godard et al. (1995) Eur. J. Biochem. 232:404-410).Preferably, microcarriers for use in ISS/MC complexes linked byhydrophobic bonding are made from hydrophobic materials, such as oildroplets or hydrophobic polymers, although hydrophilic materialsmodified to incorporate hydrophobic moieties may be utilized as well.When the microcarrier is a liposome or other liquid phase microcarriercomprising a lumen, the ISS/MC complex is formed by mixing the ISS andthe MC after preparation of the MC, in order to avoid encapsulation ofthe ISS during the MC preparation process.

Non-covalent ISS/MC complexes bound by electrostatic binding typicallyexploit the highly negative charge of the polynucleotide backbone.Accordingly, microcarriers for use in non-covalently bound ISS/MCcomplexes are generally positively charged (cationic) at physiologicalpH (e.g., about pH 6.8-7.4). The microcarrier may intrinsically possessa positive charge, but microcarriers made from compounds not normallypossessing a positive charge may be derivatized or otherwise modified tobecome positively charged (cationic). For example, the polymer used tomake the microcarrier may be derivatized to add positively chargedgroups, such as primary amines. Alternately, positively chargedcompounds may be incorporated in the formulation of the microcarrierduring manufacture (e.g., positively charged surfactants may be usedduring the manufacture of poly(lactic acid)/poly(glycolic acid)copolymers to confer a positive charge on the resulting microcarrierparticles).

As described herein, to prepare cationic microspheres, cationic lipidsor polymers, for example, 1,2-dioleoyl-1,2,3-trimethylammoniopropane(DOTAP), cetyltrimethylammonium bromide (CTAB) or polylysine, are addedeither to DP or CP, as per their solubility in these phases.

As described herein, ISS/MC complexes can be preformed by adsorptiononto cationic microspheres by incubation of polynucleotide and theparticles, preferably in an aqueous admixture. Such incubation may becarried out under any desired conditions, including ambient (room)temperature (e.g., approximately 20° C.) or under refrigeration (e.g.,4° C.). Because cationic microspheres and polynucleotides associaterelatively quickly, the incubation may be for any convenient timeperiod, such as 5, 10, 15 minutes or more, including overnight andlonger incubations. For example, ISSs can be adsorbed onto the cationicmicrospheres by overnight aqueous incubation of polynucleotide and theparticles at 4° C. However, because cationic microspheres andpolynucleotides spontaneously associate, the ISS/MC complex can beformed by sISSle co-administration of the polynucleotide and the MC.Microspheres may be characterized for size and surface charge before andafter polynucleotide association. Selected batches may then evaluatedfor activity against suitable controls in, for example, establishedhuman peripheral blood mononuclear cell (PBMC), as described herein, andmouse splenocyte assays. The formulations may also evaluated in suitableanimal models.

Non-covalent ISS/MC complexes linked by nucleotide base pairing may beproduced using conventional methodologies. Generally, base-paired ISS/MCcomplexes are produced using a microcarrier comprising a bound,preferably a covalently bound, polynucleotide (the “capturepolynucleotide”) that is at least partially complementary to the ISS.The segment of complementarity between the ISS and the capturenucleotide is preferably at least 6, 8, 10 or 15 contiguous base pairs,more preferably at least 20 contiguous base pairs. The capturenucleotide may be bound to the MC by any method known in the art, and ispreferably covalently bound to the ISS at the 5′ or 3′ end.

In other embodiments, a binding pair may be used to link the ISS and MCin an ISS/MC complex. The binding pair may be a receptor and ligand, anantibody and antigen (or epitope), or any other binding pair which bindsat high affinity (e.g., Kd less than about 10-8). One type of preferredbinding pair is biotin and streptavidin or biotin and avidin, which formvery tight complexes. When using a binding pair to mediate ISS/MCcomplex binding, the ISS is derivatized, typically by a covalentlinkage, with one member of the binding pair, and the MC is derivatizedwith the other member of the binding pair. Mixture of the twoderivatized compounds results in ISS/MC complex formation.

Many ISS/MC complex embodiments do not include an antigen, and certainembodiments exclude antigen(s) associated with the disease or disorderwhich is the object of the ISS/MC complex therapy. In furtherembodiments, the ISS is also bound to one or more antigen molecules.Antigen may be coupled with the ISS portion of an ISS/MC complex in avariety of ways, including covalent and/or non-covalent interactions, asdescribed, for example, in WO 98/16247. Alternately, the antigen may belinked to the microcarrier. The link between the antigen and the ISS inISS/MC complexes comprising an antigen bound to the ISS can be made bytechniques described herein and known in the art, including, but notlimited to, direct covalent linkage, covalent conjugation via acrosslinker moiety (which may include a spacer arm), noncovalentconjugation via a specific binding pair (e.g., biotin and avidin), andnoncovalent conjugation via electrostatic or hydrophobic bonding.

ISS Complexes with Cationic Condensing Agent and Stabilizing Agent

ISSs may be administered as a composition comprising a cationiccondensing agent, an ISS, and a stabilizing agent (i.e., CIScomposition) for modulating an immune response in the recipient. See,U.S. Patent Application No. 60/402,968. In some embodiments, the CIScomposition may also comprise an antigen and/or a fatty acid.

The CIS compositions of the invention are typically in particulate form.As will be apparent to those of skill in the art, CIS particulatecompositions of the invention will consist of a population of particlesof different sizes. Due to this naturally arising variability, the“size” of the particles in the compositions of the invention may bedescribed in ranges or as a maximum or minimum diameter. Particles areconsidered to be a particular size if at least 95% of the particles (bymass) meet the specified dimension (e.g., if at least 97% of theparticles are less than 20 μm in diameter, then the composition isconsidered to consist of particles of less than 20 μm in diameter).Particle size may be measured by any convenient method known in the art,including filtration (e.g., use of a “depth” filter to capture particlesgreater than a cutoff size), dynamic light scattering, electronmicroscopy, including TEM (particularly in combination withfreeze-fracture processing) and SEM, and the like.

Preferably, the CIS compositions of the invention comprise particleswhich are less than about 50 μm in diameter, more preferably less thanabout 20 μm in diameter, although in some embodiments the particles willbe less than about 3, 2 or 1 μm in diameter. Preferred particle sizeranges include about 0.01 μm to 50 μm, 0.02 to 20 μm, 0.05 to 5 μm, and0.05 to 3 μm in diameter.

The components of the CIS compositions may be present in variousratios/quantities in the compositions, although it is contemplated thatthe amounts of the stabilizing agent(s) and optional components such asfatty acids and antigen will remain relatively invariant, withstabilizing agents generally ranging from about 0.1% to 0.5% (v/v),fatty acids ranging from about 0 to 0.5%, and antigen concentrationsranging from about 0.1 to about 100 μg/mL, preferably about 1 to about100 μg/mL, more preferably about 10 to 50 μg/mL. The amounts and ratiosof the ISS and the cationic condensing agent are subject to a greaterrange of variation in the compositions of the invention. The amount ofISS will vary to a certain extent as a function of the molecular weightof the ISS, and generally ranges from about 50 μg/mL to about 2 mg/mL,preferably about 100 μg/mL to 1 mg/mL. The cationic condensing agent isgenerally present in excess (in terms of mass) over the ISS, generallyin ratios of about 1:2 (ISS:cationic condensing agent) to about 1:6,more preferably about 2:5 to 1:5.

Particle size in the CIS compositions is a function of a number ofvariables. The size distribution of particles in the compositions can bemodulated by altering the ratio of cationic condensing agent to ISS. Forexample, altering the ratio of cationic condensing agent to ISS in theexemplary ASS/0.4% Tween 85/0.4% oleate/polymyxin B compositions canalter mean particle size from about 1.5 μm at cationic condensingagent:IMC=1 to about 45 μm at cationic condensing agent: ISS=10.

In certain embodiments, the CIS compositions comprise a cationiccondensing agent, an ISS and a stabilizing agent that is a nonionicdetergent. In other embodiments, the compositions comprise a membranedisrupting cationic lipopeptide (preferably a polymyxin, more preferablypolymyxin B), an ISS and a stabilizing agent. In some embodiments thestabilizing agent is not a serum protein (particularly not a bovineserum protein). An exemplary composition of this class of embodimentsutilizes a polyoxyethylene ether detergent such as Tween 80 or Tween 85as the stabilizing agent, with oleate as an optional additionalstabilizing agent.

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles are made by the process of combining acationic condensing agent, an ISS and a stabilizing agent that is anonionic detergent. In other embodiments, compositions of the inventioncomprise immunomodulatory particles, wherein the particles are made bythe process of combining a membrane disrupting cationic lipopeptide(preferably a polymyxin, more preferably polymyxin B), an ISS and astabilizing agent. In some embodiments, the stabilizing agent is not aserum protein (particularly not a bovine serum protein).

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles are formed by the process of combiningan ISS and a stabilizing agent that is a nonionic detergent, therebyforming an ISS/stabilizing agent mixture, and combining a cationiccondensing agent with the ISS/stabilizing agent mixture. In otherembodiments, compositions of the invention comprise immunomodulatoryparticles, wherein the particles are formed by the process of combiningan ISS and a stabilizing agent, thereby forming an ISS/stabilizing agentmixture, and combining a membrane disrupting cationic lipopeptide(preferably a polymyxin, more preferably polymyxin B) with theISS/stabilizing agent mixture. In some embodiments, the stabilizingagent is not a serum protein (particularly not a bovine serum protein).

In some embodiments, CIS compositions comprise immunomodulatoryparticles, wherein the particles comprise a cationic condensing agent,an ISS and a stabilizing agent that is a nonionic detergent. In otherembodiments, compositions of the invention comprise immunomodulatoryparticles, wherein the particles comprise a membrane disrupting cationiclipopeptide (preferably a polymyxin, more preferably polymyxin B), anISS and a stabilizing agent. In some embodiments, the stabilizing agentis not a serum protein (particularly not a bovine serum protein).

Cationic condensing agents useful in the CIS compositions and methods ofusing the CIS compositions are molecules which are positively charged atphysiological pH (i.e., pH of about 7.0 to about 7.5). Preferably,cationic condensing agents used in the instant invention are notzwitterionic and are polycationic, that is, having more than onepositive charge per molecule. Cationic condensing agents useful in theinstant invention include hydrophilic or amphipathic polycations.

Preferred cationic condensing agents include: (a) membrane disruptingcationic lipopeptides including, but not limited to polymyxins includingpolymyxin A, polymyxin B (including polymyxin B₁ and polymyxin B₂),polymyxin C, polymyxin D, polymyxin E (also known as colistin),polymyxin K, polymyxin M, polymyxin P, polymyxin S and polymyxin T,circulins including circulin A, circulin B, circulin C, circulin D,circulin E and circulin F, octapeptin, amphotericins includingamphotericin B, and acylated peptides including octanoyl-KFFKFFKFF andacyl KALA (octanoyl-WEAKLAKALAKALAKHLAKALAKALEACEA; (b) membranedisrupting cationic peptides including, but not limited to polymyxin Bnonapeptide, cecropins including cecropin A, cecropin B and cecropin P1,KFFKFFKFF and KALA (WEAKLAKALAKALAKHLAKALAKALKACEA); (c) single chaincationic surfactants including, but not limited tocetyltrimethylammonium bromide (CTAB), benzyl-dimethyl-ammonium bromide(BDAB), CpyrB (cetyl-pyridinium bromide), CimB (cetyl imidazoliumbromide), and polycationic polymers, including, but not limited to,poly-L-lysine (PLL) and polyethyleneimine (PEI). In certain embodiments,the cationic condensing agent is a membrane disrupting cationiclipopeptide, preferably a polymyxin, more preferably polymyxin B. Insome embodiments, cationic condensing agents may exclude fatty acidesters (i.e., lipids) and double chain cationic surfactants.

Stabilizing agents useful in the CIS compositions and methods of usingthe CIS compositions include those which are suspendable in water andreduce the surface tension of water, although stabilizing agents whichare water soluble and/or completely miscible in water are preferred. Anumber of classes of stabilizing agents are useful in the compositionsand methods of the invention, including proteins (preferably hydrophilicproteins), nonionic detergents, polymeric surfactants (e.g., polyvinylalcohol and polyvinyl pyrrolidone), cationic detergents, anionicdetergents and fatty acids, although in certain embodiments, serumproteins (particularly bovine serum proteins), fatty acids, and/or ionicdetergents may be excluded from the definition of stabilizing agents.

Any protein may be used as a stabilizing agent in accordance with theinvention. In some embodiments, the stabilizing agent is a protein whichis not intended as an antigen (see discussion below); in theseembodiments, it is preferred that the protein be derived from the samespecies as the intended recipient of the composition (e.g., if thecomposition is intended for use in humans, then it is preferred that theprotein used as the stabilizing agent be a human protein). Serum albuminis an exemplary protein useful as a stabilizing agent in suchembodiments. In other embodiments, an antigen is utilizing as thestabilizing agent, in which case the antigen need not be, and in generalis preferably not, species matched with the intended recipient. Antigensuseful in the compositions and methods of the invention are disclosedbelow.

Nonionic detergents useful in the CIS compositions and methods of usingthe CIS compositions include glucamides such as decyldimethylphosphineoxide (APO-10) and dimethyldodecylphosphine oxide (APO-12),octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9)and decanoyl-N-methyl glucamide (MEGA-10), polyoxyethylene etherdetergents including polyoxyethylene(10) dodecyl ester (Genapol C100),polyoxyethylene(4) lauryl ether (BRIJ® 30), polyoxyethylene(9) laurylether (LUBROL® PX) polyoxyethylene(23) lauryl ether (BRIJ® 35),polyoxyethylene(2) cetyl ether (BRIJ® 52), polyoxyethylene(10) cetylether (BRIJ® 56), polyoxyethylene(20) cetyl ether (BRIJ® 58),polyoxyethylene(2) stearyl ether (BRIJ® 72), polyoxyethylene(10) stearylether (BRIJ® 76), polyoxyethylene(20) stearyl ether (BRIJ® 78),polyoxyethylene(100) stearyl ether (BRIJ® 700), polyoxyethylene(2) oleylether (BRIJ® 92), polyoxyethylene(10) oleyl ether (BRIJ® 97),polyoxyethylene(20) oleyl ether (BRIJ® 98),isotridecylpoly(ethyleneglycolether)₈ (Genapol 80), PLURONIC® F-68,PLURONIC® F-127, dodecylpoly(ethyleneglycolether)₉ (Thesit)polyoxyethylene(10) isooctylphenyl ether (TRITON® X-100),polyoxyethylene(8) isooctylphenyl ether (TRITON® X-114), polyethyleneglycol sorbitan monolaurate (TWEEN® 20), polyoxyethylenesorbitanmonopalmitate (TWEEN® 40), polyethylene glycol sorbitan monostearate(TWEEN® 60), polyoxyethylenesorbitan tristearate (TWEEN® 65),polyethylene glycol sorbitan monooleate (TWEEN® 80), polyoxyethylene(20)sorbitan trioleate (TWEEN® 85), poloxamer 188, andpolyethyleneglycol-p-isooctylphenyl ether (Nonidet NP40), alkylmaltoside detergents including cyclohexyl-n-ethyl-β-D-maltoside,cyclohexyl-n-hexyl-β-D-maltoside, and cyclohexyl-n-methyl-β-D-maltoside,n-decanoylsucrose, glucopyranosides including methyl6-O—(N-heptylcarbamoyl)-a-D-glucopyranoside (HECAMEG) and alkylglucopyranosides such as n-decyl-β-D-glucopyranoside,n-heptyl-β-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside,n-nonyl-β-D-glucopyranoside, n-octyl-α-D-glucopyranoside, andn-octyl-β-D-glucopyranoside, alkyl thioglucopyranosides includingn-heptyl-β-D-thioglucopyranoside, alkyl maltopyranosides includingn-decyl-β-D-maltopyranoside and n-octyl-β-D-maltopyranoside,n-decyl-β-D-thiomaltoside, digitonin, n-dodecanoyl sucrose,n-dodecyl-β-D-maltoside, heptane 1,2,3-triol,n-octanoyl-β-D-glucosylamine (NOGA), n-octanoyl sucrose, poloxamers(polyoxyethylene/polyoxypropylene block copolymers) such as poloxamer188 and poloxamer 407, and sulfobetaines including SB-10, SB-12, andSB-14 and n-undecyl-β-D-maltoside. Preferred stablizing agents includepolyoxyethylene ether detergents, particularly polyethylene glycolsorbitan monooleate and polyoxyethylene(20) sorbitan trioleate.

Anionic detergents useful in the CIS compositions and methods of usingthe CIS compositions include caprylic acid and salts thereof,chenodeoxycholic acid and salts thereof, cholic acid and salts thereof,decanesulfonic acid and salts thereof, deoxycholic acid and saltsthereof, glycodeoxycholic acid and salts thereof, lauroylsarcosine andsalts thereof, n-dodecyl sulfate and salts thereof (including sodium andlithium salts), taurochenodeoxycholic acid and salts thereof,taurocholic acid and salts thereof, taurodehydrocholic acid and saltsthereof, taurodeoxycholic acid and salts thereof, taurolithocholic acidand salts thereof, and tauroursodeoxycholic acid and salts thereof.

Cationic detergents include cetylpyridinium and salts thereof,cetyltrimethylamonia and salts thereof including cetyltrimethylammoniumbromide (CTAB), dodecyltrimethylammonia and salts thereof includingdedecyltrimethylammonium bromide, alklylammonium imidazolines,quaternary imidazolines, and tetradecyltrimtheylammonia and saltsthereof including tetradecyltrimtheylammonium bromide.

Detergents selected for use as stablizing agents are preferably thosethat are considered oil/water emulsifying detergents. Oil/wateremulsifying detergents are known in the art, and are generallycharacterized by a hydrophobic/lipophilic balance (HLB) value of about 8to about 18. Preferably, detergents incorporated into the particulatecompositions have HLB values of about 10 to about 16, more preferablyabout 11 to about 15 (e.g., polyethylene glycol sorbitan monooleate,HLB=15.4; polyoxyethylene(10) isooctylphenyl ether, HLB=13.5;polyoxyethylene(20) sorbitan trioleate HLB=11).

In certain embodiments, the CIS compositions may also include one ormore fatty acids, or a salt thereof, as an additional component. Inthose embodiments employing a fatty acid as the stablizing agentcomponent and a fatty acid as an additional component of thecomposition, the fatty acid utilized as the stablizing agent will bedifferent than the fatty acid used as the ‘additional’ component. Fattyacids useful in the CIS compositions of the invention may range in sizefrom four to 30 carbon atoms, and may be unsaturated (e.g., stearicacid), monounsaturated (e.g., oleic acid), or polyunsaturated (e.g.,linoleic acid), although monounsaturated and polyunsaturated fatty acidsare generally preferred.

In some embodiments, the CIS compositions will incorporate a fatty acidhaving a carbon chain length of at least about 4, 5, 6, 8, 10, 15, 18,or 20 carbon atoms and less than about 30, 25, 20, 19, 15 or 10 carbonatoms. Accordingly, in some embodiments, the fatty acids utilized in theinvention may have carbon chains with a length in the range of about 4to 30, 5 to 25, 10 to 20, or 15 to 20 carbon atoms.

Fatty acids useful in the CIS compositions include, but are not limitedto, arachidonic acid, decanoic acid, docosanoic acid, docosahexanoicacid eicosanoic acid, heneicosanoic acid, heptadecanoic acid, heptanoicacid, hexanoic acid, lauric acid, linoleic acid, linolenic acid,myristic acid, nonadecanoic acid, nonanoic acid, octanoic acid, oleicacid, palmitic acid, pentadecanoic acid, stearic acid, tetracosanoicacid, tricosanoic acid, tridecanoic acid, and undecanoic acid. Preferredfatty acids for use in the CIS compositions include oleic acidpalmitoleic acid, and linoleic acid.

In certain embodiments of the invention, an antigen is incorporated intothe CIS composition or administered in combination with a CIScomposition. Those CIS compositions incorporating an antigen mayincorporate the antigen into the particulate composition itself, or bedissolved or suspended in the solution in which the particulatecomposition is suspended. Any antigen may be incorporated into orco-administered with a CIS composition of the invention.

Delivery of ISS

In one embodiment, the ISS is delivered by itself into the individual.In another embodiment, the ISS is delivered with one or more antigens.In one embodiment, the antigen is co-administered with the ISS as aconjugate. In another embodiment, the antigen is administered with theISS in a separate vehicle. The administration of the antigen can becontemporaneous or simultaneous with the ISS. Discussion of delivery ofISS infra also contemplates delivery of the antigen with the ISS.

In another embodiment, the delivery of the ISS is localized to the upperrespiratory tract. As allergic rhinitis affects the nasal tract, it iscontemplated that the delivery of the ISS be localized to the nasalpassages and nasal region. As allergic rhinitis does not affect thelungs or lower respiratory tract, then care should be taken to avoidtoxicity or any other adverse effects of administering compounds thatare not necessary to treat allergic rhinitis.

ISS may be incorporated into a delivery vector, such as a plasmid,cosmid, virus or retrovirus, which may in turn code for therapeuticallybeneficial polypeptides, such as cytokines, hormones and antigens.Incorporation of ISS into such a vector does not adversely affect theiractivity.

A colloidal dispersion system may be used for targeted delivery of theISS to an inflamed tissue, such as nasal membranes. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. In one embodiment, thecolloidal system of this invention is a liposome.

Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2-4.0, um can encapsulate asubstantial percentage of an aqueous buffer containing largemacromolecules. RNA, DNA and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al, Trends Biochem. Sci., 6:77, 1981). In addition tomammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the antisense polynucleotides at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various well known linking groups can be used forjoining the lipid chains to the targeting ligand (see, e.g., Yanagawa,et al., Nuc. Acids Symp. Ser., 19:189 (1988); Grabarek, et al., Anal.Biochem., 185:131 (1990); Staros, et al., Anal. Biochem., 156:220 (1986)and Boujrad, et al., Proc. Natl. Acad. Sci. USA, 90:5728 (1993).Targeted delivery of ISS can also be achieved by conjugation of the ISSto a the surface of viral and non-viral recombinant expression vectors,to an antigen or other ligand, to a monoclonal antibody or to anymolecule which has the desired binding specificity.

Those of ordinary skill in the art will also be familiar with, or canreadily determine, methods useful in preparing oligonucleotide-peptideconjugates. Conjugation can be accomplished at either termini of the ISSor at a suitably modified base in an internal position (e.g., a cytosineor uracil). For reference, methods for conjugating oligonucleotides toproteins and to oligosaccharide moieties of Ig are known (see, e.g.,O'Shannessy, et al., J. Applied Biochem., 7:347 (1985). Another usefulreference is Kessler: “Nonradioactive Labeling Methods for NucleicAcids”, in Kricka (ed.), Nonisotopic DNA Probe Techniques (Acad. Press,1992)).

Co-administration of a peptide drug with an ISS according to theinvention may also be achieved by incorporating the ISS in cis or intrans into a recombinant expression vector (plasmid, cosmid, virus orretrovirus) which codes for any therapeutically beneficial proteindeliverable by a recombinant expression vector. If incorporation of anISS into an expression vector for use in practicing the invention isdesired, such incorporation may be accomplished using conventionaltechniques which do not require detailed explanation to one of ordinaryskill in the art. For review, however, those of ordinary skill may wishto consult Ausubel, Current Protocols in Molecular Biology, supra.

Briefly, construction of recombinant expression vectors (including thosewhich do not code for any protein and are used as carriers for ISS)employs standard ligation techniques. For analysis to confirm correctsequences in vectors constructed, the ligation mixtures may be used totransform a individual cell and successful transformants selected byantibiotic resistance where appropriate. Vectors from the transformantsare prepared, analyzed by restriction and/or sequenced by, for example,the method of Messing, et al., (Nucleic Acids Res., 9:309, 1981), themethod of Maxam, et al., (Methods in Enzymology, 65:499, 1980), or othersuitable methods which will be known to those skilled in the art. Sizeseparation of cleaved fragments is performed using conventional gelelectrophoresis as described, for example, by Maniatis, et al.,(Molecular Cloning, pp. 133-134, 1982).

Individual cells may be transformed with expression vectors and culturedin conventional nutrient media modified as is appropriate for inducingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the individual cell selected for expression, and will beapparent to the ordinarily skilled artisan.

If a recombinant expression vector is utilized as a carrier for the ISSof the invention, plasmids and cosmids are particularly preferred fortheir lack of pathogenicity. However, plasmids and cosmids are subjectto degradation in vivo more quickly than viruses and therefore may notdeliver an adequate dosage of ISS to substantially inhibit ISSimmunostimulatory activity exerted by a systemically administered genetherapy vector. Of the viral vector alternatives, adenoassociatedviruses would possess the advantage of low pathogenicity. The relativelylow capacity of adeno-associated viruses for insertion of foreign geneswould pose no problem in this context due to the relatively small sizein which ISS of the invention can be synthesized.

Other viral vectors that can be utilized in the invention includeadenovirus, adeno-associated virus, herpes virus, vaccinia or an RNAvirus such as a retrovirus. Retroviral vectors are preferablyderivatives of a marine, avian or human HIV retrovirus. Examples ofretroviral vectors in which a single foreign gene can be insertedinclude, but are not limited to: Moloney marine leukemia virus (MoMuLV),Harvey marine sarcoma virus (HaMuSV), marine mammary tumor virus(MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviralvectors can incorporate multiple genes. All of these vectors cantransfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines that have deletionsof the packaging signal include, but are not limited to, T2, PA317 andPA 12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into suchhelper cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion can be produced. By inserting one or moresequences of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector can be rendered target specific. Retroviral vectorscan be made target specific by inserting, for example, a polynucleotideencoding a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing ISS.

Pharmaceutical Compositions of ISS

If the ISS is to be delivered without use of a vector or other deliverysystem, the ISS will be prepared in a pharmaceutically acceptablecomposition. Pharmaceutically acceptable carriers preferred for use withthe ISS of the invention may include sterile aqueous of non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. Acomposition of ISS may also be lyophilized using means well known in theart, for subsequent reconstitution and use according to the invention.

Absorption promoters, detergents and chemical irritants (e.g.,keritinolytic agents) can enhance transmission of an ISS compositioninto a target tissue. For reference concerning general principlesregarding absorption promoters and detergents which have been used withsuccess in mucosal delivery of organic and peptide-based drugs, seeChien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992).

Examples of suitable nasal absorption promoters in particular are setforth at Chien, supra at Ch. 5, Tables 2 and 3; milder agents arepreferred. Suitable agents for use in the method of this invention formucosal/nasal delivery are also described in Chang, et al., Nasal DrugDelivery, “Treatise on Controlled Drug Delivery”, Ch. 9 and Table 3-4Bthereof, (Marcel Dekker, 1992). Suitable agents which are known toenhance absorption of drugs through skin are described in Sloan, Use ofSolubility Parameters from-Regular Solution Theory to DescribePartitioning-Driven Processes, Ch. 5, “Prodrugs: Topical and Ocular DrugDelivery” (Marcel Dekker, 1992), and at places elsewhere in the text.

Methods and Routes for Administration of ISS to an Individual

The ISS of the invention are administered to an individual using anyavailable method and route suitable for drug delivery. In oneembodiment, the individual is a human. In another embodiment, theindividual is a human who suffers from allergic rhinitis. In anotherembodiment, the individual is a human who has allergic rhinitis but notasthma. In another embodiment, the individual is a human who hasallergic rhinitis and allergic asthma.

One preferred method of delivery of ISS is intranasal delivery, asdescribed in the Examples. Other methods of administration include exvivo methods (e.g., delivery of cells incubated or transfected with anISS) as well as systemic or localized routes. One of ordinary skill inthe art will appreciate that methods and routes of delivery which directthe ISS into the individual should avoid degradation of the ISS in vivo.

In one aspect, the invention provides for methods of administering theISS along with an antigen that is present naturally in the environment(i.e., an adventitious antigen). Adventitious allergens can be allergensthat fluctuate in levels throughout the seasons. The presence ofseasonal allergens may be ascertained by using various sources, forexample, weather reports from weather services, news reports broadcastedby television, radio or newspaper, institutional records, and privateresearch. An example of an adventitious antigen is ragweed, e.g.,ragweed pollen allergen Antigen E (Amb a I). Other non-limiting examplesof adventitious antigens are grass allergen Lol p 1 (Tamborini et al.(1997) Eur. J. Biochem. 249:886-894), major dust mite allergens Der pIand Der pI (Chua et al. (1988) J. Exp. Med. 167:175-182; Chua et al.(1990) Int. Arch. Allergy Appl. Immunol. 91:124-129), domestic catallergen Fel d I (Rogers et al. (1993) Mol. Immunol. 30:559-568), whitebirch pollen Bet v1 (Breiteneder et al. (1989) EMBO J. 8:1935-1938),Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al. (2000)Immunology 99:625-629), and protein antigens from other tree pollen(Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl. 204:17-31).As indicated, allergens from trees are known, including allergens frombirch, juniper and Japanese cedar. Preparation of protein antigens fromgrass pollen for in vivo administration has been reported. Otherantigens that may be used are described above in Table 1.

The entrance point for many exogenous antigens into a individual isthrough the skin or mucosa. Thus, delivery methods and routes whichtarget the skin (e.g., for cutaneous and subcutaneous conditions) ormucosa (e.g., for respiratory, ocular, lingual or genital conditions)will be especially useful. Those of ordinary skill in the clinical artswill be familiar with, or can readily ascertain, means for drug deliveryinto skin and mucosa. For review, however, exemplary methods and routesof drug delivery useful in the invention are briefly discussed below.

Intranasal administration means are particularly useful in addressingrespiratory problems such as allergic rhinitis, respiratoryinflammation, particularly inflammation mediated by antigens transmittedfrom the nasal passages into the trachea or bronchioli. Such meansinclude inhalation of aerosol suspensions or insufflation of thepolynucleotide compositions of the invention. Nebulizer devices suitablefor delivery of polynucleotide compositions to the nasal mucosa, tracheaand bronchioli are well-known in the art and will therefore not bedescribed in detail here. Intranasal administration also includesspraying a liquid solution or powdered mixture instilled with thecompositions of the invention into the nose. For general review inregard to intranasal drug delivery, those of ordinary skill in the artmay wish to consult Chien, Novel Drug Delivery Systems, Ch. 5 (MarcelDekker, 1992).

Dermal routes of administration, as well as subcutaneous injections, areuseful in addressing allergic reactions and inflammation in the skin.Examples of means for delivering drugs to the skin are topicalapplication of a suitable pharmaceutical preparation, transdermaltransmission, injection and epidermal administration.

For transdermal transmission, absorption promoters or iontophoresis aresuitable methods. For review regarding such methods, those of ordinaryskill in the art may wish to consult Chien, supra at Ch. 7.Iontophoretic transmission may be accomplished using commerciallyavailable “patches” which deliver their product continuously viaelectric pulses through unbroken skin for periods of several days ormore. Use of this method allows for controlled transmission ofpharmaceutical compositions in relatively great concentrations, permitsinfusion of combination drugs and allows for contemporaneous use of anabsorption promoter.

An exemplary patch product for use in this method is the LECTRO PATCHtrademarked product of General Medical Company of Los Angeles, Calif.This product electronically maintains reservoir electrodes at neutral pHand can be adapted to provide dosages of differing concentrations, todose continuously and/or to dose periodically. Preparation and use ofthe patch should be performed according to the manufacturer's printedinstructions which accompany the LECTRO PATCH product; thoseinstructions are incorporated herein by this reference.

Epidermal administration essentially involves mechanically or chemicallyirritating the outermost layer of the epidermis sufficiently to provokean immune response to the irritant. An exemplary device for use inepidermal administration employs a multiplicity of very narrow diameter,short tynes which can be used to scratch ISS coated onto the tynes intothe skin. The device included in the MONO-VACC old tuberculin testmanufactured by Pasteur Merieux of Lyon, France is suitable for use inepidermal administration of ISS. Use of the device is according to themanufacturer's written instructions included with the device product;these instructions regarding use and administration are incorporatedherein by this reference to illustrate conventional use of the device.Similar devices which may also be used in this embodiment are thosewhich are currently used to perform allergy tests.

Systemic administration involves invasive or systemically absorbedtopical administration of pharmaceutical preparations. Topicalapplications as well as intravenous and intramuscular injections areexamples of common means for systemic administration of drugs.

Dos 1ing Parameters for ISS

A particular advantage of the ISS of the invention is their capacity toexert anti-inflammatory and/or immunotherapeutic activity even at lowdosages. Although the dosage used will vary depending on the clinicalgoals to be achieved, a suitable dosage range is one which is aneffective amount to obtain long term disease modification. In oneembodiment, the long term disease embodiment is to reduce any one of thefollowing symptoms of allergic rhinitis: nasal symptoms (rhinorrhea,congestion, excess nasal secretion/runny nose, sneezing, itching) ornon-nasal symptoms (itchy/gritty eyes, tearing, watery eyes, red orburning eyes, post-nasal drip, ear or palate itching).

In one aspect, the ISS is administered in at least 3 weekly doses. Thedosage of ISS to be administered is about 0.001 mg/kg to about 100mg/kg. In one embodiment, the dosage to be administered is 0.005 mg/kgto about 50 mg/kg. In another embodiment, dosage of ISS to beadministered is about 0.01 mg/kg to about 10 mg/kg. In anotherembodiment, at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of ISS areadministered to the individual for achieving long term diseasemodification.

The ISS is administered multiple times over a period of time. Theinterval between administration of dosages may be once a week. In thealternative, a slightly shorter period of time between administration ofdosages may be used, for example 3, 4, 5, or 6 days in betweenadministration of dosages. In another alternative, a longer period oftime may elapse in between administration of dosages, for example every8, 9, 10, 11, 12, 13, or 14 days. In yet another alternative, the ISSmay be administered in multiple dosages every 2.5 weeks, 3 weeks or 4weeks. In one embodiment, the ISS is administered in at least 3 weeklydoses at about 0.01 mg/kg to about 10 mg/kg per dose. In anotherembodiment, at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of ISS areadministered to the individual for achieving long term effect.

In yet another aspect, the ISS is administered at dosage of about 0.01mg/kg to about 10 mg/kg and at least 3 doses are administered to theindividual with about 3, 4, 5, or 6 days in between the dosages forconferring long term disease modification. In another embodiment, theISS is administered at dosage of about 0.01 mg/kg to about 10 mg/kg andat least 3 doses are administered to the individual with about 8, 9, 10,11, 12, 13, or 14 days in between the dosages for conferring long termdisease modification. In another embodiment, the ISS is administered atdosage of about 0.01 mg/kg to about 10 mg/kg and at least 3 doses areadministered to the individual with about 2.5 weeks, 3 weeks or 4 weeksin between the dosages for conferring long term disease modification. Inone embodiment, at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses of ISSare administered to the individual at intervals ranging from about 3 toabout 14 days in between dosages for achieving long term effect. Inanother embodiment, at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses ofISS are administered to the individual at intervals ranging from about2.5 weeks, 3 weeks or 4 weeks in between the dosages for achieving longterm effect. In another embodiment, these doses of ISS are administeredapproximately once a week. One of skill in the art will be able toadjust the range of dosing accordingly by measuring the levels of Th2cytokines, as exemplified in the Examples. In view of the teachingprovided by this disclosure and what is generally known at the time offiling, those of ordinary skill in the clinical arts will be familiarwith, or can readily ascertain, suitable parameters for administrationof ISS according to the invention.

In this respect, it should be noted that the anti-inflammatory andimmunotherapeutic activity of ISS in the invention is essentiallydose-dependent. Therefore, to increase ISS potency by a magnitude oftwo, each single dose should be doubled in concentration. Clinically, itmay be advisable to administer the ISS in a low dosage (e.g., about 0.01mg/kg), then increase the dosage as needed to achieve the desiredtherapeutic goal. Based on current studies, ISS are believed to havelittle or no toxicity at these dosage levels.

Kits for Use in Practicing the Methods of the Invention

For use in the methods described above, kits are also provided by theinvention. Such kits may include any or all of the following: ISS(conjugated or unconjugated); a pharmaceutically acceptable carrier (maybe pre-mixed with the ISS) or suspension base for reconstitutinglyophilized ISS; additional medicaments; a sterile vial for each ISS andadditional medicament, or a single vial for mixtures thereof, devices)for use in delivering ISS to a individual; assay reagents for detectingindicia that the immunomodulatory effects sought have been achieved intreated individuals, instructions for how to and when administer the ISSand a suitable assay device.

Examples illustrating the practice of the invention are set forth below.The examples are for purposes of reference only and should not beconstrued to limit the invention.

EXAMPLES Example 1 Inhibition of Th2-Type Gene Induction AfterIntranasal Treatment with 1018 ISS in a Mouse Model for Ragweed-InducedAllergic Asthma

One purpose of this experiment was to investigate the duration of theeffect of intranasal 1018 ISS treatment on the inhibition ofallergen-induced Th2-gene induction in ragweed-sensitized and challengedmice. The genes evaluated included various Th2-cytokines, chemokines,and various other molecules involved in airway inflammation. FemaleBALB/c mice were intraperitoneally sensitized with ragweed on Alum onday −21 and day −14. At various time points (ranging from day −7 to day0 plus 3 hrs), groups of mice were intranasally treated with 1018 ISS orsaline under light anesthesia. On day 0, all groups were challengedintranasally with either ragweed or saline. Six hrs after challenge,lungs were harvested and snap-frozen in liquid nitrogen. Total RNA wasisolated and converted into cDNA. Expression of mRNA was measured in thelung cDNA samples using real-time quantitative PCR.

The materials used were: 1018 (lot number AGU-003, Dynavax), Ragweed(Pollen lot #16, 24QQ 56-9FD-3, extract 17 Jan 03, Dynavax),pyrogenic-free saline (Sigma). The methods used were as follows: Thestudy was performed with 6-8 week old female BALB/c mice from CharlesRiver (Hollister, Calif.). A total of 90 mice were intraperitoneallysensitized with 10 μg of ragweed on Alum on day −21 and day −14.Starting from day −7 onwards groups of 5 mice were treated intranasallywith pyrogenic-free saline (50 μl) or with 1018 ISS (20 μg/50 μl saline)under light isofloraine anesthesia according to the schedule below.

sensitization day of treatment treatment challenge ragweed −7 salinesaline ragweed −7 saline ragweed ragweed −7 1018 ISS ragweed ragweed −5saline saline ragweed −5 saline ragweed ragweed −5 1018 ISS ragweedragweed −3 saline saline ragweed −3 saline ragweed ragweed −3 1018 ISSragweed ragweed −1 saline saline ragweed −1 saline ragweed ragweed −11018 ISS ragweed ragweed 0 saline saline ragweed 0 saline ragweedragweed 0 1018 ISS ragweed ragweed 0 plus 3 hrs saline saline ragweed 0plus 3 hrs saline ragweed ragweed 0 plus 3 hrs 1018 ISS ragweed

On day 0, all mice were challenged intranasally with either ragweed (5μg/50 μl saline) or saline (50 μl). Six hrs after challenge, lungs wereharvested, snap-frozen in liquid nitrogen, and stored at −80° C. forlater use. Total RNA was isolated using RNeasy mini kits (Qiagen Inc.,Valencia, Calif.). The RNA samples were DNAse-treated (RocheDiagnostics, Mannheim, Germany) and converted into cDNA usingSuperscript II Rnase H-Reverse Transcriptase (Invitrogen, Rockville,Md.) according to previously published methods (Scheerens et al., Eur.J. of Immunology 2001, 31:1465-74).

In each cDNA sample, mRNA expression levels of a variety of genes weremeasured using real-time quantitative PCR (ABI Prism 5700, Perkin ElmerApplied Biosystems) and SYBR green (Qiagen Inc., Valencia, Calif.).Sense and antisense primers used for detection were developed in houseand included primer sets to Th2-cytokines, chemokines, and various othermolecules involved in airway inflammation. In addition to the gene ofinterest, in each sample the mRNA expression of a house keeping gene wasmeasured (in this case ubiquitin). After correcting for the amount ofRNA per sample, all data were calculated relative to the expression ofthe house keeping gene (represented as gene/ubiquitin ratio).

Results: In FIG. 1, six genes essential for the development of aTh2-type airway inflammatory response are depicted and data areexpressed as gene/ubiquitin ratio. The data demonstrate that intranasalchallenge with ragweed in sensitized mice upregulated mRNA expressionlevels of Th2-genes such as IL-4, IL-5, and IL-13 when compared tosaline-challenged mice (ragweed-challenged mice denoted as RW/RW/Salinein grey bars, saline-challenged mice denoted as RW/Saline/Saline in openbars). In addition, mRNA expression levels of the chemokines TARC, MDCand eotaxin were upregulated after allergen challenge in theRW/RW/Saline mice. In contrast, in mice pretreated with 1018 ISS(denoted as RW/RW/1018 in black bars), the ragweed-induced upregulationof the various cytokines and chemokines expression levels was inhibited,however, only when 1018 ISS pretreatment was given on day −1 or on day−3, or for some genes on day −5.

In FIG. 2, gene/ubiquitin ratios are shown for GOB-5 and C2. GOB-5 andC2 (also known as FIZZ-1) are both genes that are known to be induced inthe airways by IL-4. It is clear from our data that challenge withragweed led to upregulation of both genes. In contrast, pretreatmentwith 1018 ISS given a few days before the challenge with ragweedinhibited the expression of these mRNAs associated with a Th2-typeairway inflammation. For GOB-5, 1018 ISS treatment is effective whengiven on day −1 or day −3. For C2, 1018 ISS treatment is effective whengiven on day −3 or day −5.

It has been published that pretreatment with ISS inhibitsallergen-induced airway eosinophilia and airway hyperresponsiveness in amouse model for allergic asthma (Broide et al., J. Immunol., 161:7054,1998). We have shown that this inhibition correlates with ISS-induceddown-regulation of Th2 and Th2-dependent gene expression levels in theairways (Hessel et al. (2005) J. Exp. Med., 202(11):1563).

Here, we determined the duration of ISS-mediated inhibition of theallergen-induced Th2 response in the airways. As a way to establish thiswindow of effectiveness, we measured the expression of a series of genesin the airways that are essential in or closely related to thedevelopment of Th2-type airway inflammation after allergen challenge insensitized mice. Our data demonstrate that 1018 ISS given between one tothree days before the allergen challenge is able to inhibit the majorityof these genes, which results in a greatly diminished Th2 response inthe airways. If 1018 ISS is given further removed from the allergenchallenge (i.e., earlier than day −3), we found that 1018 ISS is notable to down-regulate Th2 or Th2-dependent gene expression.

Thus, if one seeks to study the direct effects of ISS treatment on theairway Th2 response, it is advisable to pretreat within one to threedays before the allergen challenge, whereas if one is interested instudying the long-term effects of ISS on disease modification, it isadvisable to wait at least a week after ISS treatment to ensure theabsence of direct ISS effects.

Example 2 The Effects of Long-Term Intranasal Treatment with 1018 ISS ina Mouse Model for Ragweed-Induced Allergic Asthma

One purpose of this set of experiments is to investigate whetherlong-term intranasal treatment with 1018 ISS leads to diseasemodification in a mouse model for ragweed-induced allergic asthma. Thelong-term effects of weekly intranasal treatment with 1018 ISS inragweed-sensitized and challenged mice were investigated.

Mice were sensitized and subsequently challenged with an intranasal lowdose of ragweed on a weekly basis. Also on a weekly basis the mice weretreated intranasally with either saline or 1018 ISS. At several timepoints during the course of the experiment, mice were set aside to restfor a period of 2 weeks. This rest period was to ensure that the directeffects of 1018 ISS treatment had waned. At the end of the 2 weeks,these mice were re-challenged with a high dose of ragweed and theresponse to this allergen challenge was evaluated by ways of measuringthe amount of Th2 and Th1 cytokines in the airways and by determiningthe amount of airway eosinophil infiltration.

More specifically, the materials used were: 1018 (lot number AGU-003,Dynavax); Ragweed (Pollen lot #16, 24QQ 56-9FD-3, extract 17 Jan 03,Dynavax); pyrogenic-free saline (Sigma). The methods used were asfollows: The study was performed with 6-8 week old female BALB/c micefrom Charles River (Hollister, Calif.). The mice were intraperitoneallysensitized with 15 μg of ragweed on Alum on day 0 and day 7. Startingfrom day 14 onwards, mice were challenged intranasally on a weekly basiswith 0.5 μg ragweed or pyrogenic-free saline (50 μl) under lightisofloraine anesthesia. Simultaneously, the mice were treated weeklywith 1018 ISS (20 μg/50 μl saline) or pyrogenic-free saline (50 μl) viathe intranasal route. After 1, 2, 6, and after 10 weeks of antigenchallenge and ISS treatment, mice were set aside for a rest period of 2weeks and subsequently re-challenged intranasally with 5 μg of ragweed.Twenty-four hrs later lungs were lavaged and cytokines were measured inthe lavage fluid by ELISA. The detection levels for the IL-4, IL-13,IL-10, and IFN-γ ELISA were respectively 8, 8, 8, and 23 pg/ml. Lavagefluid was spun down and the cells recovered were counted using trypanblue. The remaining cells were used to prepare a cytospin and stainedwith Wright-Giemsa staining. Differential cell counts were performed andthe number of eosinophils was determined for each cytospin.

In FIGS. 3 and 4, levels of the Th2-type cytokines IL-4, IL-13 and IL-10measured in lavage fluid (BAL fluid) are depicted in pg/ml. In addition,in FIG. 4 the Th1-type cytokine IFN-γ is shown. The results indicatethat weekly challenges with ragweed in sensitized mice led to a robustTh2 inflammation in the airways with high levels of IL-4, IL-13, andIL-10, as well as a high number of eosinophils (shown in FIG. 5). Thesehigh levels of Th2 cytokines and eosinophils were absent whenragweed-sensitized mice were challenged with saline only. When mice werechallenged with ragweed and simultaneously treated with 1018 ISS, nosignificant differences were observed after 1, 2, or 6 weeks of ISStreatment when comparing ragweed-challenged mice treated with saline orwith ISS. However, after 10 weeks of 1018 ISS treatment, the Th2cytokine levels as well as the number of eosinophils were significantlydiminished (IL-13: *p<0.05; IL-4, IL-10, and eosinophils: ** p<0.01),indicating that the Th2 inflammation was inhibited in those mice.Furthermore, these data show that no increased levels of IFN-γ wereinduced in the mice treated with 1018 ISS at any of the time pointsmeasured, indicating that 10 weekly treatments with 1018 ISS did notinduce an overt Th1-type response in the airways.

Our experimental data described in this experiment demonstrated that ISStreatments did lead to disease modification, i.e. inhibition of the Th2response to allergen, however, this was in our hands not accompanied bythe development of an overt Th1 response in the airways. In Example 1,we determined that the direct effects of 1018 ISS on the Th2 response inthe airways lasted less than a week. Therefore, in this Example, allmice were rested for at least 2 weeks after their last ISS treatment,before being re-challenged with allergen. Thus any effects seen couldnot be attributed direct effects of ISS treatment. The response to there-challenge with allergen was to determine whether the airways wouldstill develop a Th2 inflammation in response to the allergen challengeor whether they had become refractory to allergen challenge. Our datashowed that at least 10 weekly intranasal 1018 ISS treatments was neededto achieve this disease modifying effect.

Example 3 The Effects of Long-Term Intranasal Treatment with 1018 ISS ina Mouse Model for Ragweed-Induced Allergic Asthma

This set of experiments was conducted to investigate whether long-termintranasal treatment with 1018 ISS conferred disease modification in amouse model for ragweed-induced allergic asthma and to evaluate whetherthis disease modification persists after 1018 ISS treatment is stoppedbut allergen exposure is continued.

Mice were sensitized and subsequently challenged with an intranasal lowdose of ragweed on a weekly basis. Also on a weekly basis the mice weretreated intranasally with either saline or 1018 ISS. At several timepoints during the course of the experiment, mice were set aside to restfor a period of 2 weeks. This rest period was to ensure that the directeffects of 1018 ISS treatment had waned. At the end of the 2 weeks,these mice were re-challenged with a high dose of ragweed and theresponse to this allergen challenge was evaluated by ways of measuringthe amount of Th2 and Th1 cytokines in the airways. The experimentalgroups included in this study were as follows:

sensitization weekly allergen weekly treatment ragweed day 0 and 7ragweed wk 1-25 saline week 1-25 ragweed day 0 and 7 ragweed wk 1-251018 ISS week 1-25 ragweed day 0 and 7 ragweed wk 1-25 1018 ISS week1-12 ragweed day 0 and 7 ragweed wk 1-12 1018 ISS week 1-12

The purpose of the mice receiving ISS treatment for 12 weeks andallergen challenges for a total of 25 weeks was to evaluate whetherISS-induced disease modification was long-lasting in the presence ofcontinued allergen exposure.

More specifically, the materials used were: 1018 (lot number AGU-003,Dynavax); Ragweed (Pollen lot # Jan. 26, 2005, Dynavax); pyrogenic-freesaline (Sigma). The study was performed with 6-8 week old female BALB/cmice from Charles River (Hollister, Calif.). The mice wereintraperitoneally sensitized with 15 μg of ragweed on Alum on day 0 andday 7. Starting from day 14 onwards, mice were challenged intranasallyon a weekly basis with 0.5 μg ragweed or pyrogenic-free saline (50 μl)under light isofloraine anesthesia. Simultaneously, the mice weretreated weekly with 1018 ISS (20 μg/50 μl saline) or TOLAMBA (20 μg/50μl saline) or pyrogenic-free saline (50 μl) via the intranasal route.After 1, 8, 12, 16, and after 25 weeks of antigen challenges and ISStreatment, mice were set aside for a rest period of 2 weeks andsubsequently re-challenged intranasally with 5 μg of ragweed.Twenty-four hours later lungs were lavaged and cytokines were measuredin the lavage fluid by ELISA.

Results: In FIG. 6, levels of the Th2-type cytokines IL-4, IL-5, IL-13and IL-10 measured in lavage fluid (BAL fluid) are depicted in pg/ml.The detection levels for the IL-4, IL-5, IL-13, IL-10, and IFN-γ ELISAwere respectively 8, 8, 8, 8, and 23 pg/ml. In addition, the Th1-typecytokine IFN-γ was measured but no induction of IFN-γ above detectionlevel was measured in any of the treatment groups. Our results show thatweekly challenges with ragweed in sensitized mice led to a robust Th2inflammation in the airways with high levels of IL-4, IL-5, IL-13, andIL-10. When mice were challenged with ragweed and simultaneously treatedwith 1018 ISS, no significant differences were observed after 1 week ofISS treatment when comparing ragweed-challenged mice treated with salineor with ISS. However, after 8, 12, 16, and 25 weeks of 1018 ISStreatment, the Th2 cytokine levels were significantly diminished,indicating that the allergen-induced Th2 inflammation was inhibited inthe 1018 ISS-treated mice. The observation that no detectable levels ofIFN-γ were induced in the mice treated with 1018 ISS at any of the timepoints measured indicates that 25 weekly treatments with 1018 ISS didnot induce an overt Th1-type response in the airways. In groups treatedfor 12 weeks with 1018 ISS that subsequently continued to receiveallergen challenges for another 13 weeks, the Th2 response remainedinhibited, indicating that the disease modification induced by 1018 ISSis long-lasting.

In Example 2, we demonstrated that 10 weekly ISS treatments led todisease modification, i.e., inhibition of the Th2 response to allergen,however, this was not accompanied by the development of an overt Th1response in the airways. The experiment described here extends thisfinding with the observation that disease modification is in factalready achieved after 8 weekly 1018 ISS treatments and that thisdisease modification persists even when allergen exposures continued foranother 13 weeks.

Example 4 ISS-Conjugates

Methods similar to Example 3 above were used except that both 1018 ISSand 1018 ISS conjugated to Amb a I (conjugate known as TOLAMBA) wereused. FIG. 7 is a graph that depicts the results of measuring Th2-typecytokines, IL-4, IL-5, IL-10, and IL-13 in mice that have been treatedwith 20 μg of 1018 ISS via the intranasal route on a weekly basis for 25weeks or 20 μg TOLAMBA via the intranasal route on a weekly basis for 25weeks. Th2 suppression was also observed when using ISS-conjugate.Epidemiological studies have consistently shown that allergic asthma andrhinitis often coexist in the same patients (1,2). Both airway diseasesshare the same trends of increasing incidence (3), predisposing factors(2), and pathophysiological mechanisms following allergen encounter(4,5) and benefit from treatment with topical steroids (6). Inliterature successful measurement of allergic rhinitis has beendescribed in ovalbumin-sensitized and challenged mice (7). In this studythe ovalbumin challenges were given by ways of an aerosol, and therebyaffecting both the lungs and nasal passages. The study referred toincludes nasal lavage eosinophil counts and measurement of the thicknessof the nasal mucosa.

Example 5 The Effects of Long-Term Intranasal Treatment with 1018 ISS ina Mouse Model for Ragweed-Induced Allergic Rhinitis

Epidemiological studies have consistently shown that allergic asthma andrhinitis often coexist in the same patients (Parikh, A. et al., Br. Med.J. (1997) 314:1392-5, and Lundback B., Clin. Exp Allergy (1998)28:3-10). Both airway diseases share the same trends of increasingincidence (Aberg, N et al., Clin. Exp. Allergy (1995) 25:815-9),predisposing factors (Lundback, supra), and pathophysiologicalmechanisms following allergen encounter (Durham, S R., Clin. Exp.Allergy (1998) 28:11-16, and Chanez, P. et al., Am. J. Respir. Crit.Care Med. (1999) 159:588-95) and benefit from treatment with topicalsteroids (Welsch, P W et al., Mayo Clin. Proc. (1987) 62:125-34). In theliterature, successful measurement of allergic rhinitis has beendescribed in ovalbumin-sensitized and challenged mice (Hellings, P W etal., Clin. Exp. Allergy, (2001) 31: 782-790). In that study theovalbumin challenges were given by ways of an aerosol, and therebyaffecting both the lungs and nasal passages. That study referred toincludes nasal lavage eosinophil counts and measurement of the thicknessof the nasal mucosa.

In this Example, experiments as described in Examples 3 and 4 arerepeated and nasal parameters reflective of allergic rhinitis areevaluated. The number of weekly 1018 ISS or 1018 ISS-conjugatetreatments is varied as well as the length of the period in which weeklyallergen exposures are continued after cessation of the 1018 ISS or 1018ISS-conjugate treatment. Nasal parameters include eosinophil counts andIL-4, IL-5, IL-13, IL-10 and IFN-g cytokine measurements in the nasallavage. In addition, ISS-inducible gene expression is evaluatedalongside with the evaluation of gene expression levels reflective ofTh2 inflammation. Histological analysis of the nasal passage and morespecifically of the nasal mucosa is included.

With the establishment of disease modification, two questions arose: 1)Can disease modification be reversed after re-sensitizing the animals?2) Is the disease modifying effect on the Th2 response antigen-specific?To answer these questions, mice were sensitized with ragweed,subsequently weekly challenged for 13 weeks with ragweed, and eithertreated or not with 1018 ISS. This was followed by a period of 4 weeklyintranasal ragweed challenges. This regimen has been proven to inducedisease modification in prior experiments. From there, animals eitherrested or went through a re-sensitizing phase with either ragweed andalum, or with ovalbumin and alum (2 intraperitoneal injections separatedby a week). One week after the last injection, all mice received a finalintranasal antigen challenge. A naïve, age-matched control group wasused to demonstrate that animals of the same age could be successfullysensitized to either ragweed or ovalbumin.

Results: After a 13-week treatment course with 1018 ISS inragweed-sensitized and challenged mice, the responsiveness of theairways to a high-dose ragweed challenge is silenced. Subsequentsystemic re-sensitization with ragweed or ovalbumin followed by anairway allergen challenge (with ragweed or ovalbumin respectively) doesnot lead to airway eosinophilia or elevated bronchoalveolar (BAL) Th2cytokines (FIGS. 8 & 9 respectively). This response to re-sensitizationis the same, regardless of whether re-sensitization is performed withragweed or ovalbumin (the “same” allergen or a “different” allergen).The presence of ovalbumin-specific IgE antibodies in the serum ofISS-treated groups (FIG. 10) indicates that the systemic sensitizationwith ovalbumin was successful. This data implies that the localenvironment in the airways has changed as a consequence of ISS-treatmentand can prevent an allergen-induced Th2 response to a newly introducedallergen, even if the allergen was not present during the period thatthe ISS treatment was given.

1. A method for treating an individual in need thereof comprisingadministering to the individual multiple administrations of an effectiveamount of an ISS wherein the administration results in a long termdisease modification of allergic rhinitis.
 2. The method of claim 1wherein the multiple administrations occur on a weekly basis.
 3. Themethod of claim 1 wherein the ISS is administered at least 3 times overthe course of the treatment.
 4. The method of claim 1 wherein the ISS isadministered four or more times.
 5. The method of claim 1 wherein thelong term disease modification is a decrease of Th2 response in theindividual.
 6. The method of claim 5 wherein the decrease of Th2response in the individual is a decrease of any one of the cytokinesselected from group consisting of IL-4, IL-5, IL-10, and IL-13.
 7. Themethod of claim 1 wherein the long term disease modification lasts atleast 13 weeks after the last administration of the ISS.
 8. The methodof claim 1 wherein the individual in need thereof has allergic rhinitis.9. The method of claim 1 wherein the ISS is selected from the groupconsisting of 1018 ISS, a CpG containing ISS, and a chimericimmunomodulatory compound.
 10. The method of claim 1 wherein the ISS is1018 ISS.
 11. The method of claim 10 wherein the ISS is administered inthe presence of an allergen.
 12. The method of claim 11 wherein theallergen is an adventitious allergen.
 13. The method of claim 12 whereinthe adventitious allergen is ragweed.
 14. The method of claim 11 whereinthe allergen is a seasonal allergen.
 15. The method of claim 1 whereinthe individual is a human with allergic rhinitis but not asthma.